Enantioselective oligomerization of alpha-hydroxy carboxylic acids and alpha-amino acids

ABSTRACT

An enzymatic synthesis and composition of oligomers and co-oligomers comprised of α-hydroxy carboxylic acids and α-amino acids or peptides is disclosed. In a preferred embodiment, a α-hydroxy carboxylic acid with a specific chiral configuration is linked by an amide linkage to a α-amino acid specific with a specific chiral configuration or linked by an amide linkage to a peptide made up of α-amino acid monomers having identical chiral configurations. Proteolytic enzymes catalyze oligomerization of the α-hydroxy carboxylic acid and α-amino acid. The degree and distribution of oligomerization varies upon the type and concentrations of different reaction mixtures utilized and upon the length of allowed reaction time. The resultant oligomers may be provided to animals such as ruminants as bioavailable amino acid supplements that are resistant to degradation in the rumen and other animals such as swine, poultry and aquatic animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/288,196, filed May 2, 2001, and as acontinuation-in-part of U.S. patent application Ser. No. 09/699,946,filed Oct. 30, 2000, which claims priority from U.S. ProvisionalApplication Serial No. 60/162,725, filed Oct. 29, 1999 (now abandoned).The entire texts of U.S. Provisional Application Serial No. 60/288,196,U.S. patent application Ser. No. 09/699,946 and U.S. ProvisionalApplication Serial No. 60/288,196 are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a process for theenantioselective preparation of oligomers consisting of α-amino acidisomers and co-oligomers consisting of α-hydroxy carboxylic acid isomersand α-amino acid isomers. The present invention also relates tocompositions containing such oligomers and co-oligomers and methods ofuse thereof.

[0003] In an effort to improve nutrition, the diets of ruminant animalshave been supplemented with proteins and naturally occurring α-aminoacids. Unfortunately, these proteins and α-amino acids can be subjectedto extensive degradation in the rumen by ruminal microorganisms, therebyrendering the protein or amino acid unavailable to the animal forabsorption. This is not a very efficient utilization of the feed, whichis especially problematic in animals having increased nutritionalrequirements such as lactating dairy cows and fast growing animals suchas beef cattle.

[0004] One approach to solving this problem has been to modify orprotect the dietary protein or amino acid by a variety of chemical andphysical methods so that it escapes degradation in the rumen. Forexample, heating soybean meal has shown some promise in producingprotected proteins. However, the results were highly variable.Underheating the protein resulted in no protection while overheating theprotein resulted in the degradation of important essential amino acids.See, for example, Plegge, S. D., Berger, L. L. and Fahey Jr. G. C. 1982.Effect of Roasting on Utilization of Soybean Meal by Ruminants. J. Anim.Sci. 55:395 and Faldet, M. A., Son, Y. S. and Satter, L. D. 1992.Chemical, in vitro and in vivo evaluation of soybean heat-treated byvarious processing methods. J. Dairy Sci. 75:789. Similarly, physicalcoating of proteins with materials such as fats and calcium soaps offats has been met with mixed success.

[0005] Therefore, there is a need to somehow protect the protein fromdegradation in the rumen in order to make it available to the animal inthe intestine where it can be properly absorbed. This would allow theanimal to get increased nutritional benefit from the feed. Increasingthe nutritional benefit of the feed can reduce the amount of feedrequired by the animals.

[0006] Dietary supplements such as proteins, naturally occurring α-aminoacids, vitamins, minerals, and other nutrients are also used inaquaculture, (i.e., the cultivation of aquatic animals such as fish andcrustaceans). Many of such supplements are difficult to provide,however, due to being soluble in water which causes the supplements todissolve before they can be ingested. Dietary supplements for use inaquaculture therefore are preferably in an insoluble form in order to beingested.

[0007] The role played by short chain peptides and their derivatives inthe areas of nutrition science, flavor chemistry, and pharmacology hasprimed the advances in peptide chemistry. The inherent advantages ofenzymatic peptide synthesis has led to its evolution as an alternativeto chemical coupling methods (Fruton, J. S., 1992, Adv. Enzymology, 53,239-306). The thiol-protease papain is reported to be the most efficientcatalyst for aqueous phase synthesis of homooligomers of hydrophobicamino acids like leucine, methionine, phenylalanine, and tyrosine (A.Ferjancic, A. Puigserver and H.Gaertner, Biotech. Lett, 13(3) (1991)161-166). The equilibria of such reactions is tilted in favor ofsynthesis by the precipitation of hydrophobic oligomers. However, thedifficulty involved in the analysis of higher order, water insolubleoligomers, presents a unique challenge to biochromatography.

SUMMARY OF THE INVENTION

[0008] Among the objects of the present invention, therefore, is theprovision of an oligomer which is protected from degradation in therumen of a ruminant, the provision of such an oligomer which providesnutritional or pharmacological benefit to the animal, and the provisionof a process for the preparation of such oligomers.

[0009] A further object of the present invention is the provision of aco-oligomer and oligomer that provides nutritional or pharmacologicalbenefit to animals, and the provision of a process for the preparationof such co-oligomers and oligomers.

[0010] A further object of the invention is the provision of aco-oligomeric or oligomeric coating for vitamins, minerals, ornutrients.

[0011] Another object of the present invention is the provision of amethod to purify enantiomeric mixtures of α-hydroxy carboxylic acids,α-amino acids, or combinations thereof.

[0012] Briefly, therefore, the present invention is directed to aprocess for the preparation of an oligomer consisting of α-amino acidisomers. The process comprises forming a reaction mixture containing (i)an enzyme and (ii) an an enantiomeric mixture of α-amino acid, orderivative thereof. An oligomer is formed that incorporates oneenantiomer of the enantiomeric mixture of the α-amino acid or derivativethereof in preference to the other enantiomer.

[0013] The present invention is further directed to a compositioncomprising a residue of an α-hydroxy carboxylic acid bonded to a peptideby an amide or an ester linkage, said peptide comprising two or moreα-amino acid residues, each of said α-amino acids being independentlyselected from the group consisting of α-amino acids. Preferably, morethan 50% of the α-amino acid residues in the peptide are of identicalchirality.

[0014] The present invention is further directed to an oligomer of theformula CA-(AA)_(n)-wherein CA is the residue of an α-hydroxy carboxylicacid, each AA is the residue of an α-amino acid or derivative thereofwherein greater than one-half of the AA residues are derived from thegroup consisting of α-amino acids or derivatives thereof having the samechiral configuration, and n is at least 2.

[0015] The present invention is also directed to a process for providingan animal with a food ration. The process comprises providing anoligomer or a co-oligomer prepared from a mixture containing an enzyme,an α-amino acid, and optionally, an α-hydroxy carboxylic acid orderivative thereof. The feed ration is administered to the animal byoral administration, eye spray, placement in ear, placement in nasalcavity, and bucchal administration, sublingual administration, rectaladministration or injection.

[0016] The present invention is further directed to an orallyadministered dietary supplement comprising a vitamin, mineral, ornutrient that is coated with an oligomeric coating. The coatingcomprises a residue of an α-hydroxy carboxylic acid bonded to a peptideby an amide linkage. The peptide comprises two or more independentα-amino acids independently selected from the group consisting ofα-amino acids.

[0017] The present invention is further directed to a process forproviding an animal with a dietary supplement comprising a vitamin,mineral, or nutrient. The process comprises coating the vitamin, mineralor nutrient with a composition to form a dietary supplement andadministering the dietary supplement to the animal. The compositioncomprises a residue of an α-hydroxy carboxylic acid bonded to a peptideby an amide linkage and the peptide comprises two or more independentα-amino acids independently selected from the group consisting ofα-amino acids.

[0018] The present invention is further directed to a process forpurifying an enantiomeric mixture of α-amino acid or derivative thereof.The process comprises forming a reaction mixture comprising (i) anenzyme, (ii) an enantiomeric mixture of α-amino acid or a derivativethereof, and (iii) an α-hydroxy carboxylic acid or a derivative thereof.A peptide reaction product is formed from the reaction mixturecomprising (i) an oligomer or co-oligomer from the combination whichincorporates one of the members of the enantiomeric mixture of α-aminoacid or derivative thereof in preference to a second enantiomer of theenantiomeric mixture, and (ii) unreacted second enantiomer. The oligomeror co-oligomer and unreacted second enantiomer are then separated fromthe reaction product and each other.

[0019] The present invention is also directed to a process for purifyingan α-hydroxy carboxylic acid enantiomer or derivative thereof in anenantiomeric mixture. The process comprises forming a reaction mixturecomprising (i)an enzyme, (ii) an enantiomeric mixture of an α-hydroxycarboxylic acid and (iii) an α-amino acid or a derivative thereof. Areaction product is formed from the reaction mixture comprising (i) aco-oligomer which preferentially incorporates a first enantiomer over asecond enantiomer of the enantiomeric mixture, and (ii)unreacted secondenantiomer. The co-oligomer and unreacted second enantiomer are thenseparated from the reaction product and each other.

[0020] Other objects and features of this invention will be in partapparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a MALDI-TOF graph of methionine oligomers andco-oligomers from a papain catalyzed synthesis.

[0022]FIG. 2 is a MALDI-TOF graph of HMB-methionine co-oligomers from apapain catalyzed synthesis.

[0023]FIG. 3 is a HPLC graph of methionine sulfone oligomers.

[0024]FIG. 4 is a HPLC graph of HMB-methionine sulfone co-oligomersafter a incubation period of 10 minutes.

[0025]FIG. 5 is a HPLC graph of HMB-methionine sulfone co-oligomersafter a incubation period of 24 hours.

[0026]FIG. 6 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a reverse micellarsystem.

[0027]FIG. 7 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a reversemicellar system.

[0028]FIG. 8 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a 2-phasesystem.

[0029]FIG. 9 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a 2-phase system.

[0030]FIG. 10 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a 3-phase system.

[0031]FIG. 11 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a 3-phasesystem.

[0032]FIG. 12 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a reduced volume2-phase system.

[0033]FIG. 13 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a reducedvolume 2-phase system.

[0034]FIG. 14A is a chromatogram of persulfonated methionine oligomersusing a UV absorption detector.

[0035]FIG. 14B is a positive ion total ion chromatogram of persulfonatedmethionine oligomers.

[0036]FIG. 15A is a chromatogram of persulfonated HMB-methionineco-oligomers using a UV absorption detector.

[0037]FIG. 15B is a positive ion total ion chromatogram of persulfonatedHMB-methionine co-oligomers.

[0038]FIG. 16 is a positive ion ESI spectra of (Met)₃ sulfone peakeluting at 5.27 minutes.

[0039]FIG. 17 is a positive ion ESI spectra of (Met)₄ sulfone peakeluting at 7.70 minutes.

[0040]FIG. 18 is a positive ion ESI spectra of (Met)₅ sulfone peakeluting at 9.47 minutes.

[0041]FIG. 19 is a positive ion ESI spectra of (Met)₆ sulfone peakeluting at 11.09 minutes.

[0042]FIG. 20A is a positive ion ESI spectra of (Met)₇ sulfone peakeluting at 12.7 minutes.

[0043]FIG. 20B is a positive ion ESI spectra of (Met)₈ sulfone peakeluting at 14.26 minutes.

[0044]FIG. 20C is a positive ion ESI spectra of (Met)₉ sulfone peakeluting at 15.60 minutes.

[0045]FIG. 21A is a chromatogram of persulfonated methionine oligomersusing a UV absorption detector.

[0046]FIG. 21B is a total ion chromatogram ESI-negative ion ofpersulfonated methionine oligomers.

[0047]FIG. 22A is a chromatogram of persulfonated HMB-methionineco-oligomers using a UV absorption detector.

[0048]FIG. 22B is a total ion chromatogram ESI-negative ion ofpersulfonated HMB-methionine co-oligomers.

[0049]FIG. 23 is a negative ion ESI spectra of HMB-(Met)₅ sulfone peakeluting at 11.57 minutes.

[0050]FIG. 24 is a negative ion ESI spectra of HMB-(Met)₆ sulfone peakeluting at 13.86 minutes.

[0051]FIG. 25 is a negative ion ESI spectra of HMB-(Met)₇ sulfone peakeluting at 15.31 minutes.

[0052]FIG. 26 is a bar graph of the relative distribution of (Met)_(n)wherein n is the number of methionine residues in the methionineoligomers.

[0053]FIG. 27 is a bar graph of the relative distribution ofHMB-(Met)_(n) wherein n is the number of methionine residues in theHMB-methionine co-oligomers.

[0054]FIG. 28A is a positive ion ESI-MS spectra of HMB-methionineco-oligomers synthesized with HMB methyl ester and methionine ethylester.

[0055]FIG. 28B is a negative ion ESI-MS spectra HMB-methionineco-oligomers synthesized with HMB methyl ester and methionine ethylester.

[0056]FIG. 29 is a parent ion SSI-MS spectra HMB-methionine co-oligomerssynthesized with HMB methyl ester and methionine ethyl ester.

[0057]FIG. 30 is a daughter ion spectrum of (Met)₆-ethyl ester.

[0058]FIG. 31A is a positive ion ESI-MS spectra of tyrosine (Tyr)noligomers wherein n is the number of tyrosine residues in the oligomers.

[0059]FIG. 31B is a negative ion ESI-MS spectra of tyrosine (Tyr)noligomers wherein n is the number of tyrosine residues in the oligomers.

[0060]FIG. 32A is a positive ion spectra of HMB-tyrosine co-oligomers.

[0061]FIG. 32B is a negative ion spectra of HMB-tyrosine co-oligomers.

[0062]FIG. 33A is a positive ion ESI-MS spectra of leucine oligomers.

[0063]FIG. 33B is a negative ion ESI-MS spectra of leucine oligomers.

[0064]FIG. 34A is a positive ion ESI-MS spectra of HMB-leucineco-oligomers.

[0065]FIG. 34B is a negative ion ESI-MS spectra of HMB-leucineco-oligomers.

[0066]FIG. 35A is a positive ion ESI-MS spectra of HMB-phenylanalineco-oligomers.

[0067]FIG. 35B is a negative ion ESI-MS spectra of HMB-phenylanalineco-oligomers.

[0068]FIG. 36 is a graph of the effect of Aqueous: Non-Aqueous ratios on(Lys)_(n) oligomer yield wherein n is the number of lysine residues inthe oligomers in a two-phase system.

[0069]FIG. 37 is a bar graph of the effect of volumetric ratios on thedegree of (Lys)_(n) oligomer yield in a two-phase reaction systemwherein n is the number of lysine residues in the oligomers.

[0070]FIG. 38 is a graph of the effect of additive concentrations on(Lys)_(n) oligomer yield wherein n is the number of lysine residues inthe oligomers.

[0071]FIG. 39 is a bar graph of the effect of additive concentrations onthe degree of (Lys)_(n) oligomerization wherein n is the number oflysine residues in the oligomers.

[0072]FIG. 40 is a graph of the effect of substrate concentrations on(Lys)_(n) oligomer yield wherein n is the number of lysine residues inthe oligomers.

[0073]FIG. 41 is a bar graph of the distribution of lysine oligomersformed in reaction mixtures with varied substrate concentrations.

[0074]FIG. 42 is a graph of the effect of incubation time on totallysine oligomer yield.

[0075]FIG. 43 is a bar graph of the distribution of lysine oligomersformed after different incubation time periods.

[0076]FIG. 44 is a graph of the effect of aqueous to non-aqueous solventphase ratios on total lysine oligomer yield in a three-phase system.

[0077]FIG. 45 is a bar graph of the distribution of lysine oligomersformed in reaction mixtures at various aqueous to non-aqueous solventratios.

[0078]FIG. 46 is a graph of the effect of additive concentrations on thetotal lysine oligomers yield.

[0079]FIG. 47 is a bar graph of the distribution of lysine oligomersformed with varied additive concentrations in a 2-phase system.

[0080]FIG. 48 is a graph of the total lysine oligomer yield formed afterdifferent incubation time periods in a 3-phase system.

[0081]FIG. 49 is a bar graph of the distribution of lysine oligomersformed after a one hour incubation period in a 3-phase system.

[0082]FIG. 50 is a bar graph of the distribution of lysine oligomersformed after a 24 hour incubation period in a 3-phase system.

[0083]FIG. 51 is a chromatogram of an enantiomeric mixture of methionineethyl ester using a UV absorption diode array detector (DAD).

[0084]FIG. 52 is a chromatogram of a enantiomeric mixture of methionineethyl ester and HMB-ethyl ester using a UV absorption diode arraydetector (DAD).

[0085]FIG. 53 is a chromatogram of an oligomer and co-oligomerhydrolyzate illustrating the presence of only the L-methionineenantiomer using a UV absorption diode array detector (DAD).

[0086]FIG. 54 is a chromatogram of an oligomer and co-oligomerhydrolyzate illustrating the presence of only the L-HMB enantiomer usinga UV absorption diode array detector (DAD).

[0087]FIG. 55A is a positive ion ESI-MS spectra of the lacticacid—methionine oligomers prepared in Example 15.

[0088]FIG. 55B is a negative ion ESI-MS spectra of the lacticacid—methionine oligomers prepared in Example 15.

[0089]FIG. 56A is a positive ion ESI-MS spectra of the lacticacid—tyrosine oligomers prepared in Example 15.

[0090]FIG. 56B is a negative ion ESI-MS spectra of the lacticacid—tyrosine oligomers prepared in Example 15.

[0091]FIG. 57A is a positive ion ESI-MS spectra of the lacticacid—leucine oligomers prepared in Example 15.

[0092]FIG. 57B is a negative ion ESI-MS spectra of the lacticacid—leucine oligomers prepared in Example 15.

[0093]FIG. 58A is a positive ion ESI-MS spectra of the lacticacid—tryptophan oligomers prepared in Example 15.

[0094]FIG. 58B is a negative ion ESI-MS spectra of the lacticacid—tryptophan oligomers prepared in Example 15.

[0095]FIG. 59A is a positive ion ESI-MS spectra of the lacticacid—phenylalanine oligomers prepared in Example 15.

[0096]FIG. 59B is a negative ion ESI-MS spectra of the lacticacid—phenylalanine oligomers prepared in Example 15.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] In accordance with the present invention, it has been discoveredthat oligomers and co-oligomers of α-hydroxy carboxylic acids andα-amino acids may be prepared in an enzymatically catalyzed reaction.

[0098] The α-hydroxy carboxylic acid/α-amino acid oligomersenzymatically synthesized by the process of the present invention maypossess altered properties from those of the α-amino acid monomers. Forexample, it has been suggested that the α-hydroxy carboxylicacid/α-amino acid oligomers, unlike proteins, peptides, or amino acidmonomers, are not recognized in the rumen by ruminal microorganisms. Asa result, the ruminal microorganisms do not break down the oligomers andthe oligomers are available for absorption by the ruminant.

[0099] Further, the solubility properties of many of the α-hydroxycarboxylic acid/α-amino acid oligomers of the present invention are alsodifferent from their monomeric counterpart. Thus, while many α-aminoacid monomers, such as methionine, are soluble in water, the α-hydroxycarboxylic acid/α-amino acid oligomers and α-amino acid oligomers formedfrom methionine monomers are insoluble. This alteration advantageouslypermits the oligomers to be introduced in aqueous environments withoutbeing dissolved in the solution.

[0100] In general, the oligomers of the present invention comprise theresidue of an α-hydroxy carboxylic acid bonded to the residue of anα-amino acid by an amide or an ester linkage. Thus, the oligomerscorrespond to the general formula CA-(AA)_(n) wherein CA comprises theresidue of an α-hydroxy carboxylic acid, (AA)_(n) is an oligomericsegment comprising the residue of one or more independent α-amino acids,n is at least 1 and CA is bonded to (AA)_(n) by an amide or an esterlinkage. In accordance with a preferred embodiment, the α-hydroxycarboxylic acid is bonded to the residue of an α-amino acid with anamide bond to effectively create an α-amino acid oligomer that is“end-capped” by an α-hydroxy carboxylic acid residue.

[0101] If the reaction mixture does not contain an α-hydroxy carboxylicacid, an α-amino acid oligomer is formed that corresponds to the formula(AA)_(n) wherein each AA is the residue of one or more independentα-amino acids, n is at least 2 and the amino acid residues are bonded toeach other by an amide linkage or an ester.

[0102] It is important to note that when n is greater than 1, (AA)_(n)may comprise more than one independent α-amino acid residue. Statedanother way, (AA)_(n) comprises a peptide comprising two or moreindependent α-amino acids. Thus, the composition of the oligomer may beadvantageously tailored for specific applications. For example, in apreferred embodiment, the oligomer may be designed to meet the essentialamino acid requirements of a particular animal by incorporating two ormore different amino acid residues (e.g., methionine and lysineresidues) into an oligomer. Such an example is an oligomer comprisinglysine and methionine residues in a 3:1 ratio, which would meet theessential amino acid requirements of a ruminant.

[0103] Several variables affect the value of n, such as the amino acidmonomer utilized, the reaction solution composition, and the method ofisolating the co-oligomer products. Typically, n is less than 20. Insome embodiments, n ranges from about 1 to about 10, more typically fromabout 2 to about 8 and, in some embodiments, from about 3 to about 5.For example, in oligomers comprising methionine residues, n typicallyranges from about 4 to about 12, with an average of about 6 to about 8.

[0104] The oligomers of the present invention may be obtained (and used)as a dimer, trimer, tetramer, pentamer, hexamer, septamer, octamer,nonamer, decamer, etc. in which a residue of the α-hydroxy carboxylicacid is linked to a residue of an α-amino acid via an amide or esterlinkage. Alternatively, an oligomeric segment may be obtained which ischemically or enzymatically linked to another moiety, for example,through the α-hydroxy group of the α-hydroxy carboxylic acid residue,the carboxy terminus of the α-amino acid residue (for oligomerscomprising an amide linkage between the α-hydroxy carboxylic acidresidue and the α-amino acid residue) or the amino terminus of theα-amino acid residue (for oligomers comprising an ester linkage betweenthe α-hydroxy carboxylic acid residue and the α-amino acid residue).

[0105] In a preferred embodiment, the oligomer or oligomeric segmentcorresponds to the structure:

[0106] R¹ is hydrogen, hydrocarbyl or substituted hydrocarbyl,

[0107] R² is hydrogen, hydrocarbyl, substituted hydrocarbyl, or ahydroxy protecting group,

[0108] R³ is hydrogen, hydrocarbyl or substituted hydrocarbyl,

[0109] each AA is the residue of an α-amino acid selected from the groupconsisting of α-amino acids independently of any other α-amino acidresidue, and

[0110] n is at least 1.

[0111] α-Hydroxy Carboxylic Acid Residue

[0112] In general, the oligomer or oligomeric segments of the presentinvention may comprise the residue of any α-hydroxy carboxylic acid.Preferred α-hydroxy carboxylic acids correspond to the general structureR¹R³C(OR²)COOH wherein R¹ is hydrogen, hydrocarbyl or substitutedhydrocarbyl; R² is hydrogen, a hydroxy protecting group, hydrocarbyl, orsubstituted hydrocarbyl; and R³ is hydrogen, hydrocarbyl or substitutedhydrocarbyl, preferably hydrogen. For example, the α-hydroxy carboxylicresidue may be the residue of any of the following naturally occurringα-hydroxy carboxylic acids (with R¹ for such acid being given inbrackets): lactic acid [—CH₃], mandelic acid [—C₆H₅], malic acid[—CH₂COOH], and tartaric acid [—CH(OH)COOH]. In addition, the α-hydroxycarboxylic acid residue may be the residue of an α-hydroxy acid analogof a naturally occurring α-amino acid, more preferably the residue ofthe α-hydroxy analog of an essential α-amino acid, and still morepreferably the residue of the α-hydroxy analog of methionine, i.e.,2-hydroxy-4-(methylthio)butyric acid.

[0113] In general, the α-hydroxy carboxylic acid residue may comprisethe residue of an α-hydroxy carboxylic acid having the D- configuration,the L-configuration, or a racemic or other mixture of the D- andL-isomers. In some embodiments, however, it is generally preferred thatthe α-hydroxy carboxylic acid residue comprise the residue of anα-hydroxy carboxylic acid having the L-configuration.

[0114] Further, it is important to note that the α-hydroxy carboxylicacid residue incorporated into the oligomer may comprise the residue ofmore than one α-hydroxy carboxylic acid. Thus, the residue may comprisea homo-oligomer containing one or more α-hydroxy carboxylic acidmonomers or a hetero-oligomer containing two or more independentα-hydroxy carboxylic acid monomers.

[0115] α-Amino Acid Residue(s)

[0116] In general, the oligomers of the present invention may comprisethe residue of any α-amino acid. Preferred α-amino acids correspond tothe general structure R^(a)R^(b)C(NH₂)COOH wherein R^(a) is hydrogen,hydrocarbyl, substituted hydrocarbyl or heterocyclo; and R^(b) ishydrogen. For example, the α-hydroxy amino residue(s) may be theresidue(s) of any of the naturally occurring α-amino acids, e.g.,asparagine, glycine, alanine, valine, leucine, isoleucine,phenylalanine, proline, serine, threonine, cysteine, methionine,tryptophan, tyrosine, glutamine, aspartic acid, glutamic acid, lysine,arginine, and histidine. Preferably, the α-amino acid residue(s) includethe residue(s) of one or more essential α-amino acids, i.e., isoleucine,phenylalanine, leucine, lysine, methionine, threonine, tryptophan,histidine and valine. Still more preferably, the α-amino acid residue(s)include the residue(s) of methionine and/or lysine.

[0117] In general, the α-amino acid residue may comprise the residue ofan α-amino acid having the D-configuration, the L-configuration, or aracemic or other mixture of the D- and L-isomers. In some embodiments,however, it is generally preferred that the α-amino acid residuecomprise the residue of an α-amino acid having the L-configuration.

[0118] Further, it is important to note that the α-amino acid residueincorporated into the oligomer may comprise the residue of more than oneα-amino acid. Thus, the residue may comprise a homo-oligomer containingone or more α-amino acid monomers or a hetero-oligomer containing two ormore independent α-amino acid monomers.

[0119] Enzymatic Oligomerization

[0120] The oligomers of the present invention are enzymaticallysynthesized in a mixture. The mixture comprises at least one α-hydroxycarboxylic acid or a derivative thereof, at least one α-amino acid or aderivative thereof, and an enzyme.

[0121] The α-hydroxy carboxylic acid may be present in the mixture as afree acid or as a carboxylic acid derivative, e.g., the correspondingester, acid halide, amide, anhydride, or ketene. Preferably, theα-hydroxy carboxylic acid and its derivatives have the formulaR¹R³C(OR²)COY or R¹RC(OR²)═C═O wherein R¹, R² and R³ are as previouslydefined and Y is hydroxy (for the free acid), halogen (for acid halidederivatives), hydrocarbyloxy (for ester derivatives), amino (for amidederivatives), and hydrocarbylcarboxy (for anhydride derivatives). Insome embodiments, the α-hydroxy carboxylic acid is preferably present inthe mixture in the form of an ester, i.e., where Y is —OR⁵ and R⁵ ishydrocarbyl, more preferably alkyl, alkene, or aryl, still morepreferably lower alkyl. In other embodiments, the α-hydroxy carboxylicacid is preferably present in the mixture in the form of an amide, i.e.,where Y is —NR⁶R⁷ and R⁶ and R⁷ are independently hydrogen orhydrocarbyl, more preferably lower alkyl, still more preferablyhydrogen.

[0122] The mixture may contain more than one α-hydroxy carboxylic acidspecies. Thus, for example, the mixture may contain the hydroxy analogof methionine (in one or more of its free acid, acid halide, amide,anhydride or ketene forms) and, in addition, one or more other α-hydroxycarboxylic acids such as lactic acid, mandelic acid, malic acid, ortartaric acid (in one or more of their respective free acid, acidhalide, amide, anhydride or ketene forms).

[0123] In addition to, or instead of α-hydroxy carboxylic acid monomers,the mixture may further contain oligomers (e.g., dimers, trimers,tetramers, pentamer, hexamer, septamer, octamer, nonamer, decamer, etc.)of one or more α-hydroxy carboxylic acids. For example, the mixture maycontain a homo-oligomer formed from HMB or another α-hydroxy carboxylicacid or a hetero-oligomer of an α-hydroxy carboxylic acid (e.g., HMB)and at least one other α-hydroxy carboxylic acid.

[0124] Similarly, the α-amino acids may be present in the mixture as afree acid or as a carboxylic acid derivative, e.g., the correspondingester, acid halide, amide, anhydride, or ketene. In general, the α-aminoacid and its derivatives have the formula R^(a)R^(b)C(NH₂)COY orR^(a)C(OR²)═C═O wherein R^(a), R² and R^(b) are as previously definedand Y is hydroxy (for the free acid), halogen (for acid halidederivatives), hydrocarbyloxy (for ester derivatives), amino (for amidederivatives), and hydrocarbylcarboxy (for anhydride derivatives). Insome embodiments, the α-amino acid is preferably present in the mixturein the form of an ester, i.e., where Y is —OR⁵ and R⁵ is hydrocarbyl,more preferably alkyl or aryl, still more preferably lower alkyl. Inother embodiments, the α-amino acid is preferably present in the mixturein the form of an amide, i.e., where Y is —NR⁶R⁷ and R⁶ and R⁷ areindependently hydrogen or hydrocarbyl, more preferably lower alkyl,still more preferably hydrogen.

[0125] The mixture may contain more than one α-amino acid species. Thus,for example, the mixture may contain one α-amino acid (in one or more ofits free acid, acid halide, amide, anhydride or ketene forms) and, inaddition, one or more other α-amino acids (in one or more of theirrespective free acid, acid halide, amide, anhydride or ketene forms). Byway of further example, the mixture may contain methionine (in one ormore of its free acid, acid halide, amide, anhydride or ketene forms)and, in addition, one or more other nutritionally important α-aminoacid(s) such as lysine, tryptophan and/or phenylalanine (in one or moreof their respective free acid, acid halide, amide, anhydride or keteneforms).

[0126] In addition to, or instead of α-amino acid monomers, the mixturemay contain oligomers (e.g., dimers, trimers, tetramers, pentamer,hexamer, septamer, octamer, nonamer, decamer, etc.) of one or moreα-amino acids. For example, the mixture may contain a homo-oligomerformed from methionine, lysine or other α-amino acid or ahetero-oligomer of an α-amino acid (e.g., methionine) and at least oneother nutritionally important α-amino acid such as lysine, tryptophanand/or phenylalanine.

[0127] The reaction mixture further comprises an enzyme. The enzyme maybe dissolved in the mixture or, alternatively, it may be adsorbed orotherwise immobilized onto a variety of substrates. For example, theenzyme may be immobilized onto controlled pore glass, agarose,sepharose, nylon, or polyethylene glycol. Enzymes may also be adsorbed,for example, onto activated charcoal, ion exchange resins, silica,polyacrylamide, collagen, starch, bentonite, ultramembrane filters,cellulose, alumina, titania, and polyvinylchloride. In addition, enzymesmay be retained by entrapment, microencapsulation, liposome formation,hollow fiber, inorganic bridge formation, and aggregation.

[0128] The type of enzyme selected will determine the direction that anoligomerization process proceeds. For example, enzymes generallycharacterized as a protease when included in a reaction mixture alongwith, for example, an α-hydroxy carboxylic acid ethyl ester and anα-amino acid ethyl ester, will cause a peptide reaction product to beformed from the reaction mixture. The peptide reaction product comprisesan oligomer comprising α-amino acids and the α-hydroxy carboxylic acidbonded together by amide bonds. Examples of suitable protease enzymesinclude serine proteinases (e.g., Trypsin, α-Chymotrypsin, Elastase,Carboxypeptidase, and Subtilisin), thiol proteinases (e.g., Papain,Ficin, Bromelain, Streptococcal proteinase, Cathepsins, Calpains,Clostripain, and Actinidin), metalloproteinases (e.g., Thermolysin),acid proteinases (e.g., Pepsin, Penicillopepsin, Chymosin, Cathepsin,and Renin), liver esterase (e.g., pig liver esterase), alkalineprotease, carbonic anhydrase, nonribosomal peptide synthetase, thrombin,cardosins A or B, or pronase.

[0129] If, however, an enzyme such as a lipase enzyme is used, thereaction mixture containing the lipase enzyme, an α-hydroxy carboxylicacid, and an α-amino acid or derivative thereof, instead forms apolyester reaction product. Enantioselective lipase enzymes may beobtained from a variety of microorganisms such as Candida cylindracea,Candida lipolytica, Candida antarctica (bacteria) and fungi such asRhizopus oryzae, Aspergillus niger, and the like. The reaction productwill therefore comprise an oligomer wherein the α-amino acids and theα-hydroxy carboxylic acid are bonded together by ester bonds. If thereaction mixture comprises a lipase enzyme and an ester of an α-hydroxycarboxylic acid or a derivative thereof, an oligomer of α-hydroxycarboxylic acid will form wherein the monomers are linked together byester bonds.

[0130] In a preferred embodiment, the mixture contains an enzyme whichcatalyzes the formation of peptide bonds. Exemplary enzymes includeserine proteinases (e.g., Trypsin, α-Chymotrypsin, Elastase,Carboxypeptidase, and Subtilisin), thiol proteinases (e.g., Papain,Ficin, Bromelain, Streptococcal proteinase, Cathepsins, Calpains,Clostripain, and Actinidin), metalloproteinases (e.g., Thermolysin),acid proteinases (e.g., Pepsin, Penicillopepsin, Chymosin, Cathepsin,and Renin), liver esterase (e.g., pig liver esterase), alkalineprotease, carbonic anhydrase, nonribosomal peptide synthetase, thrombin,cardosins A or B, or pronase.

[0131] Enantioselective Enzymatic Oligomerization

[0132] It has further been found that the present invention may beutilized to enzymatically synthesize oligomers, co-oligomers, orsegments thereof, consisting of α-hydroxy carboxylic acid isomers andα-amino acid isomers or α-amino acid isomers wherein one enantiomer ofthe α-hydroxy carboxylic acids, α-amino acids, or derivatives thereof isincorporated into the co-oligomer or oligomer in preference to the otherenantiomer. Stated another way, it has been found that by using anenantioselective enzyme in the process of the present invention, peptideor ester reaction products comprising co-oligomers, oligomers orsegments thereof can be formed from a reaction mixture comprising anenantiomeric mixture of α-hydroxy carboxylic acids, α-amino acids, orderivatives thereof, wherein one enantiomer of the enantiomeric mixtureis incorporated into the reaction product in preference to the otherenantiomer of the mixture.

[0133] As previously described for enzymatic oligomerization, theoligomers of the present invention are enantioselectively synthesized ina mixture. The mixture comprises at least one at least one α-amino acidor a derivative thereof as described above, an enantioselective enzyme;and, optionally a α-hydroxy carboxylic acid or a derivative thereof asdescribed above.

[0134] The α-hydroxy carboxylic acids and α-amino acids may be presentin the mixture as enantiomeric mixtures. An enantiomeric mixturecontains enantiomeric pairs of the α-hydroxy carboxylic acids, α-aminoacids, or derivatives thereof. The proportion of each species may varyfrom a racemic mixture that contains equal proportions of the D- andL-isomer configurations (e.g., 50% of the L-isomer and 50% of theD-isomer), to enantiomeric mixtures wherein one species isproportionally greater than its opposite species (e.g., an enantiomericmixture containing 70% L-isomer and 30% D-isomer).

[0135] In one embodiment of the present invention, the reaction mixturecontains a racemic mixture of α-amino acid. In another embodiment, thereaction mixture contains a racemic mixture of α-hydroxy carboxylicacid. In still another embodiment, the reaction mixture contains racemicmixtures of both α-hydroxy carboxylic acid and α-amino acid.

[0136] In general, the mixture contains an enzyme whichenantioselectively catalyzes the formation of peptide bonds betweenα-amino acids having identical chiral configurations (e.g., L-isomers ofamino acids). Thus, the co-oligomer or oligomer formed from the mixturecomprises a residue of an α-hydroxy carboxylic acid bonded to a peptideby an amide linkage or an ester linkage, wherein the peptide comprisestwo or more independent α-amino acid residues having identical chiralconfiguration.

[0137] In another embodiment, the enzyme further enantioselectivelycatalyzes the formation of the amide or ester linkage between theα-hydroxy carboxylic acid residue and the α-amino acid residue such thatthe oligomer comprises one α-hydroxy carboxylic acid enantiomer linkedto the α-amino acid oligomer in preference to another α-hydroxycarboxylic acid enantiomer. For example, a reaction mixture containingpapain, an enantiomeric mixture of methionine ethyl ester isomers and anenantiomeric mixture of HMB ethyl ester isomers will form oligomersconsisting of L-HMB linked to one or more L-methionine residues. Theenantiospecificity of the enzyme is dependent upon the α-amino acid andα-hydroxy carboxylic acid being oligomerized. Exemplary enantioselectiveenzymes include thiol proteinases (e.g., Papain, Bromelain, Cathepsin s,Cathepsin b, and Cathepsin c) and serine proteinases (e.g., some formsof Subtilisin). In a preferred embodiment, the enantioselective enzymeenzymatically links the carboxy terminus of the α-hydroxy carboxylicacid to the amino terminus of the α-amino acid.

[0138] It is important to note that either of the α-hydroxy carboxylicacid residue or the α-amino acid residues may comprise an oligomer(i.e., a dimer, trimer, etc.) as described above and still beenantioselectively incorporated into the oligomers of the presentinvention. For example, if the enantioselective enzyme in the reactionmixture is suitable for incorporating oligomers comprising theL-enantiomer, the enzyme will catalyze the oligomerization of anyα-hydroxy carboxylic acid residue or α-amino acid residue having anL-configuration.

[0139] The enantioselective enzyme may be dissolved in the mixture or,alternatively, it may be adsorbed or otherwise immobilized onto avariety of substrates. For example, the enzyme may be immobilized ontocontrolled pore glass, agarose, sepharose, nylon, or polyethyleneglycol. Enantioselective enzymes may also be adsorbed, for example, ontoactivated charcoal, ion exchange resins, silica, polyacrylamide,collagen, starch, bentonite, ultramembrane filters, cellulose, alumina,titania, and polyvinylchloride. In addition, enzymes may be retained byentrapment, microencapsulation, liposome formation, hollow fiber,inorganic bridge formation, and aggregation.

[0140] In one embodiment, the oligomer formed from the enantioselectiveoligomerization comprises a composition comprising a residue of anα-hydroxy carboxylic acid bonded to a peptide by an amide or an esterlinkage, wherein the peptide comprises two or more α-amino acid residuesand each of the α-amino acids of the peptide are independently selectedfrom the group consisting of α-amino acids. Preferably, more than 50% ofthe α-amino acid residues in the peptide are of identical chirality and,more preferably, essentially all of the α-amino acid residues in thepeptide are of identical chirality.

[0141] In a preferred embodiment, the oligomer or oligomeric segmentformed by an enantioselective enzyme corresponds to the structure:

[0142] wherein,

[0143] R¹ is hydrogen, hydrocarbyl or substituted hydrocarbyl,

[0144] R² is hydrogen, hydrocarbyl, substituted hydrocarbyl, or ahydroxy protecting group,

[0145] R³ is hydrogen, hydrocarbyl or substituted hydrocarbyl,

[0146] each AA is the residue of an α-amino acid or derivative thereofwherein greater than one-half of the AA residues are derived fromα-amino acids or derivatives thereof having the same chiralconfiguration, and

[0147] n is at least 2.

[0148] For example, a mixture of papain, an enantiomeric mixture of HMBethyl ester, and an enantiomeric mixture of D, L-methionine ethyl esterin a reaction mixture has been found to form homo-oligomers ofL-methionine and hetero-oligomers of L-HMB/L-methionine wherein theL-HMB is linked through its carboxy terminus to the amino terminus ofthe methionine oligomer. The homo-oligomers and hetero-oligomers may beseparated out of the solution by precipitation, filtration, selectiveextraction, column chromatography, lyophilization, and evaporationtechniques. Typically, an oligomer comprising about nine methionineamino acids will precipitate out of solution and may be easily filteredor centrifuged away from the reacting mixture containing the freehydroxy acids and α-amino acids. Soluble methionine oligomers comprisedof lower numbers of methionine residues can be separated from the freeamino and hydroxy acids using membrane filtration. Once the reaction isallowed to run to near completion, the remaining reaction mixturecontains papain, a significant amount of monomers of D-HMB ethyl esterand D-methionine ethyl ester (e.g., about 95% of the total amount of HMBethyl ester and methionine ethyl ester isomers), and a small amount ofL-HMB ethyl ester and L-methionine ethyl ester. Papain may be removedfrom the solution by size exclusion chromatography or similar separationtechnique known in the art. The remaining monomers of D-HMB ethyl esterand D-methionine ethyl ester may be removed from solution by rotaryevaporation for purification or transformed to their respective L-isomerform through base racemization and recycled.

[0149] If the reaction mixture does not contain an α-hydroxy carboxylicacid, but does contain an enantioselective enzyme and an α-amino acid orderivative thereof, an α-amino acid oligomer is formed that correspondsto the formula (AA)_(n). AA is the residue of an α-amino acid comprisingtwo or more independent α-amino acids wherein greater than one-half ofthe AA residues are derived from α-amino acids or derivatives thereofhaving the same chiral configuration, n is at least 2, and the aminoacid residues are bonded to each other by an amide linkage. Typically, nwill be less than 20. In some embodiments, n will range from about 2 toabout 10, more typically from about 2 to about 8 and, in someembodiments, from about 3 to about 5. In methionine oligomers, forexample, n typically will range from about 4 to 12, with an average of 6to 8.

[0150] Enzymatic Reaction Mixtures

[0151] In one embodiment of the present invention, the enzymaticreaction is carried out in a single phase, aqueous solution underconditions typically employed in enzyme catalyzed reactions for thepreparation of oligomers and co-oligomers of α-amino acids. Such systemsare typically used in enzymatic biochemical reaction. See, e.g.,Lehninger, Nelson, and Cox, Principles of Biochemistry, 1993, WorthPublisher, NY, N.Y.

[0152] In a second embodiment of the present invention, the enzymaticreaction is carried out in a two-phase system comprising an aqueousphase and an organic phase. In general, the organic phase comprises anorganic solvent selected from the group consisting of alkanes, alkenes,aryls and suitable derivatives thereof. See, e.g., Olmsted and Williams,Chemistry the Molecular Science, 1994, Mosby Publisher, St. Louis, Mo.

[0153] In a third embodiment of the present invention, the enzymaticreaction is carried out in a reverse micelle system. Such a systemcomprises a continuous organic phase, a dispersed aqueous phase, and asurfactant to obtain and stabilize micelle phase. In general, theorganic phase comprises an organic solvent selected from the groupconsisting of alkyl, aryl, and suitable derivatives thereof, and thesurfactant is selected from the group consisting of ionic or non-ionicsurfactants. Such reverse micelle systems are typically used forbiotechnological reactions. See, e.g., Vicente, Aires-Barros, and Empis,J. Chem. Tech. Biotechnol. 1994, 60, 291.

[0154] In a fourth embodiment of the present invention, the enzymaticreaction is carried out in a three-phase system comprising an aqueousphase, a first organic phase and a second organic phase with the twoorganic phases being immiscible. In general, the first organic phasecomprises an organic solvent selected from the group consisting ofhydrocarbon solvents and the second organic phase comprises an organicsolvent selected from the group consisting of halogenated hydrocarbon,perhalogenated hydrocarbon, and halogenated hydrocarbyl solvents. Suchthree phase systems are routinely used for chemical and biochemicalreactions.

[0155] In general, the reaction may be carried out over a relativelywide range of temperatures, e.g., about 4° C. to about 50° C., typicallyabout 35 to about 40° C. The pH of the aqueous phase is typically about5.5 to about 9. Depending upon whether the reaction is carried out in asingle phase, aqueous solution or in a multi-phase system, the ratio ofthe water phase to the organic phase may range from 100:0 to 0.1:99.9parts by weight, respectively. Reaction time may varying from minutes tohours (e.g., from about 10 minutes to about 24 hours or more) dependingon the desired yield and the synthesis may be achieved both with andwithout physical agitation of the reaction mixture.

[0156] Separation

[0157] Specific oligomers and co-oligomers can be separated from thereaction mixtures through precipitation, filtration, selectiveextraction, column chromatography, lyophilization, and evaporationtechniques. Often, the oligomeric and co-oligomeric products areprecipitates which may be easily filtered or centrifuged away from thepeptide and ester reaction product mixture containing free hydroxy acidsand unreacted α-amino acids. For example, soluble oligomer andco-oligomeric products can be separated from reaction product mixtureusing membrane filtration. Alternatively, free amino acids and α-hydroxyacids may be removed from the product mixture using ion exchange orother applicable chromatographic technique. The selection of separationprocedure is dependent on the desired oligomers and co-oligomers.

[0158] When enantioselective enzymes are utilized to form co-oligomersconsisting of α-hydroxy carboxylic acid isomers and α-amino acid isomershaving identical chiral configurations from a reaction mixturecontaining a racemic mixture of α-hydroxy carboxylic acid isomers and aracemic mixture of α-amino acids, the reaction mixture after thereaction is formed will contain the enzyme and a greater proportion ofthe non-selected enantiomers of α-hydroxy carboxylic acid and α-aminoacid than the non-selected enantiomers. The enzyme may be removed fromsolution by filtration and recycled, thereby leaving a solutionprimarily containing monomers of the non-selected enantiomers. Further,the non-selected enantiomers may be separated from the solution byrotary evaporation or by other methods known in the art. Afterseparation, the non-selected enantiomers may be transformed into themonomeric form of the selected isomer through base racemization andrecycled. For example, when the L-isomer of methionine is oligomerizedor co-oligomerized by papain in a reaction mixture containing a racemicmixture of methionine and HMB, at the end of the reaction, the mixturewill primarily be comprised of papain, D-methionine, and D-HMB. Papainmay be simply filtered from the reaction mixture by size exclusionchromatography leaving a solution primarily containing D-methionine(e.g., approximately 95% or more of the remaining racemic mixture ofmethionine) which may be isolated by rotary evaporation.

[0159] Alternatively, the process of the present invention can be usedto recover the selected enantiomer from the separated oligomer. Forexample, the recovered oligomer may be hydrolyzed with acid to separatethe first, selected enantiomer or derivative thereof from otherhydrolyzates. The separated enantiomer may then be racemized andrecycled for further use.

[0160] Uses

[0161] Biological systems such as ruminants, poultry, swine, and aquaticanimals readily absorb and utilize the L-isomers of amino acids but areunable to utilize the corresponding D-isomer without first transformingthe D-isomer into the L-isomer through an oxidation followed bytransamination enzymatic reactions. As such reactions require additionaltime and energy to be expended by the animal before the amino acids canbe utilization by the animal, feed supplements of L-isomer oligomers andco-oligomers are advantageous as they can be utilized by the animal withminimal expenditure of energy, which ultimately improves the growth rateof the animals.

[0162] The enantioselective oligomerization and co-oligomerization ofspecific enantiomers of α-hydroxy carboxylic acids, α-amino acids, orderivatives thereof results in the purification of the species in theoriginal enantiomeric mixtures. First, by selectively oligomerizing orco-oligomerizing enantiomeric species, such as the L-enantiomers, theresulting oligomers and co-oligomers formed are therefore a more pureform of L-enantiomers. The L-enantiomers may then be isolated throughaddition of acid to the oligomers and co-oligomers thereby hydrolyzingthem into the L-enantiomers with which they are comprised. The L-isomersmay be further isolated from each other through chromatographic or otherseparation means known in the art.

[0163] Conversely, while the L-isomers are being selectivelyoligomerized and co-oligomerized, the reaction mixture contains anincreasingly greater proportion of the non-selected enantiomer species,for example, the D-enantiomers. Thus, as the reaction progresses, theremaining unreacted enantiomers, such as D-α-hydroxy carboxylic acids,D-α-amino acids, or derivatives thereof, may be recovered from thereaction mixture by rotary evaporation or other means. The D-isomers ofα-hydroxy carboxylic acids, α-amino acids, or derivatives thereof maythen be transformed to their respective L-isomer form through baseracemization and reused as reactants in additional oligomerization andco-oligomerization reactions.

[0164] Depending upon the desired application, the α-hydroxy carboxylicacid/α-amino acid co-oligomer and α-amino acid oligomer compositions ofthe present invention may be provided to animals as an amino acidsupplement. The α-hydroxy carboxylic acid/α-amino acid co-oligomer andα-amino acid oligomer compositions may be fed or otherwise administeredorally, or sprayed into the eye, ear or nasal cavity of an animal,preferably a ruminant. Alternatively, the compositions may be injected,or administered bucchally (i.e., to the gums), sublingually (i.e.,beneath the tongue) or rectally.

[0165] The α-hydroxy carboxylic acid/α-amino acid co-oligomercompositions of the present invention or α-amino acid oligomercompositions enzymatically formed in the absence of an α-hydroxycarboxylic acid may also be used to supplement the diets of animals notpossessing rumens, such as poultry, swine, and aquatic animals. As withruminants, these compositions may be fed or otherwise administeredorally, bucchally, sublingually, rectally, sprayed into the eye, ear ornasal cavity of an animal, or injected into the animal.

[0166] These compositions may be further used in aquaculture by applyingthe compositions to an aquatic habitat in a particle size that is ableto be ingested by the target animal. The compositions may be used inaquaculture as particles of the α-amino acid oligomer or α-hydroxycarboxylic acid/α-amino acid co-oligomer itself or as an ingredient inthe animal's feed rations. While oligomers and co-oligomers may beutilized in varying sizes, feed mills typically manufacture feedsupplements in particle sizes of about 0.25 mm or more for incorporationinto feed rations. The feed ration pellets containing the oligomer orco-oligomer ingredient would be sized according to the animal to whichit is being fed. For example, fish, such as carp and trout, may be fedthe oligomer or co-oligomer compositions as an ingredient of a feedration that is applied to the surface of the water. Feed mills typicallyproduce fish feed rations in particle sizes that range between about 4mm to about 5 mm in diameter. Conversely, for smaller aquatic animals,such as shrimp, that cannot ingest large particles due to small mouthsize, the compositions may be applied to the surface of the water aspure forms of the oligomer or co-oligomer or as ingredients of a feedration in smaller feed particle sizes. Feed mills typically producerations for smaller aquatic animals in particles that are at least 1.6mm in diameter, preferably between about 2 mm to about 3 mm in diameter,more preferably between about 2.2 mm to about 2.4 mm in diameter. Thelength of the feed ration pellets are typically manufactured to be twoto three times the length of the diameter. While feed mills may producefeed rations in particular size ranges, the dimensions of the rationthat incorporates the oligomer or co-oligomer composition may be variedtherefrom without diminishing the effectiveness of the oligomer orco-oligomer.

[0167] In another embodiment, the α-hydroxy carboxylic acid/α-amino acidco-oligomer or α-amino acid oligomer compositions may be used as aprotective coating for vitamins, minerals, and other nutrientsupplements that are ingested by both humans and other animals, forexample, ruminants, poultry, swine, and aquatic animals. Vitamins andother nutritional supplements (e.g., vitamin A, acetate or palmitateester, and the like) which are ingested often must be protected againstacids and proteolytic enzymes present in the stomach and rumen in orderto be available for absorption by the animal in the intestine. Thesesupplements may often also be soluble in water or sensitive to oxidationsuch that they cannot remain in a solid form that can be ingested andutilized in an aqueous environment. Currently, protective coatings, suchas fat and gelatin based coatings, are applied to vitamins and nutrientsto protect against their degradation in the stomach or rumen ordissolution in water. These coatings may be made from animal productssuch as beef fat and gelatin. Such sources have recently come underscrutiny due to potential diseases carried by the animals which mayaffect the availability and quality of fats and gelatin used forcoatings.

[0168] The α-hydroxy carboxylic acid/α-amino acid co-oligomer or α-aminoacid oligomer compositions provide a superior alternative toanimal-based coatings. As some oligomer or co-oligomer compositions maybe resistant to degradation in the stomach and rumen, as well asinsoluble in water, they may be used as vitamins, minerals, and othernutrient coatings. Thus, in ruminants, the co-oligomer compositions maycoat supplements in order for the vitamins, minerals, and othernutrients to bypass microbial degradation that occurs in the rumen. Oncein the small intestine, the co-oligomer compositions are completelydegraded wherein the ruminant may absorb the α-hydroxy carboxylic acids,amino acids, and the previously coated vitamins, minerals, andnutrients. Once absorbed, the ruminant may convert the α-hydroxycarboxylic acids from the coating to its respective amino acid forutilization by the ruminant. Since the α-hydroxy carboxylic acid/α-aminoacid co-oligomer and α-amino acid oligomer coatings are enzymaticallysynthesized, they do not introduce the risk of infecting the ruminantwith a disease that may have been carried by an animal from which a fator gelatin based coating is derived.

[0169] For non-ruminants, gastric acids and enzymes present in thestomach begin to degrade the coating as it passes through the stomach tothe intestine. Once in the intestine, the coating is completelydegraded, and the previously encapsulated vitamins, minerals, and othernutrients may be absorbed.

[0170] Some supplements, such as vitamin A, are soluble in water. Leftuncoated, the soluble supplements would dissolve into the surroundingwater and pollute it rather than provide the aquatic animals with thevitamins, minerals, and other nutrient supplements they need. As theα-hydroxy carboxylic acid/α-amino acid co-oligomer or α-amino acidoligomer compositions are also insoluble in water, they also may bebeneficially used to coat vitamins, minerals and other nutrientsupplements for use in aquaculture.

[0171] The application of α-hydroxy carboxylic acid/α-amino acidco-oligomer or α-amino acid oligomer coatings to vitamins and othernutritional supplements may be achieved by methods known in the art. Forexample, the oligomer or co-oligomer may be dissolved in a volatilesolvent and subsequently spray coated on a fluidized bed of thesupplements. As the solvent evaporates, a coating of α-hydroxycarboxylic acid/α-amino acid co-oligomer or α-amino acid oligomerremains on the supplements which may then be provided to the animal.

[0172] Definitions

[0173] The term “aquaculture” refers to the cultivation of aquaticanimals including, but not limited to, freshwater and salt water fish(e.g., carp, trout, catfish, bass, sea bass, cod, salmon, and fishrelated thereto) and crustaceans (e.g., shrimp, crabs, lobster,freshwater shrimp, and the like).

[0174] The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Preferably, thesemoieties comprise 1 to 20 carbon atoms.

[0175] The “substituted hydrocarbyl” moieties described herein arehydrocarbyl moieties which are substituted with at least one atom otherthan carbon, including moieties in which a carbon chain atom issubstituted with a hetero atom such as nitrogen, oxygen, silicon,phosphorous, boron, sulfur, or a halogen atom. These substituentsinclude halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,hydroxy, protected hydroxy, keto, acyl, acyloxy; nitro, amino, amido,nitro, cyano, and thiol.

[0176] The alkyl groups described herein are preferably lower alkylcontaining from one to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includemethyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.

[0177] The alkenyl groups described herein are preferably lower alkenylcontaining from two to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includeethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and thelike.

[0178] The alkynyl groups described herein are preferably lower alkynylcontaining from two to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includeethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

[0179] The terms “aryl” or “ar” as used herein alone or as part ofanother group denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Phenyl andsubstituted phenyl are the more preferred aryl.

[0180] The terms “halogen” or “halo” as used herein alone or as part ofanother group refer to chlorine, bromine, fluorine, and iodine.

[0181] The terms “heterocyclo” or “heterocyclic” as used herein alone oras part of another group denote optionally substituted, fully saturatedor unsaturated, monocyclic or bicyclic, aromatic or nonaromatichydrocarbon groups having at least one heteroatom in at least one ring,and preferably 5 or 6 atoms in each ring. The heterocyclo grouppreferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4nitrogen atoms in the ring, and may be bonded to the remainder of themolecule through a carbon or heteroatom. Exemplary heterocyclo includefuryl, thienyl, pyridyl and the like. Exemplary substituents include oneor more of the following groups: hydrocarbyl, substituted hydrocarbyl,keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy,alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, and thiol.

[0182] The acyl moieties described herein contain hydrocarbyl,substituted hydrocarbyl or heterocyclo moieties.

[0183] The terms “hydroxyl protecting group” and “hydroxy protectinggroup” as used herein denote a group capable of protecting a freehydroxyl group (“protected hydroxyl”) which, subsequent to the reactionfor which protection is employed, may be removed without disturbing theremainder of the molecule. A variety of protecting groups for thehydroxyl group and the synthesis thereof may be found in “ProtectiveGroups in Organic Synthesis” by T. W. Greene, John Wiley and Sons, 1981,or Fieser & Fieser. Exemplary hydroxyl protecting groups include acetyl(Ac), benzyl (PhCH₂—), 1-ethoxyethyl (EE), methoxymethyl (MOM),(methoxyethoxy)methyl (MEM), (p-methoxyphenyl)methoxymethyl (MPM),tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBPS),tert-butoxycarbonyl (Boc), tetrahydropyranyl (THP), triphenylmethyl(Trityl, Tr), 2-methoxy-2-methylpropyl, benzyloxycarbonyl (Cbz),trichloroacetyl (OCCCl₃), benzyloxymethyl (BOM), tert-butyl (t-Bu),triethylsilyl (TES), trimethylsilyl (TMS), and triisopropylsilyl (TIPS).

[0184] The abbreviation “HMB” shall mean the 2-hydroxy analog ofmethionine, i.e., 2-hydroxy-4-(methylthio)butyric acid.

[0185] The terms “chiral,” “chiral configuration,” and “enantiomer”refer to a particular stereoisomer of a molecule. For example,L-methionine and L-HMB.

[0186] The term “identical chirality” or “identical chiralconfiguration” refers to the chiral carbon of two or more moleculeshaving the same stereoisomeric configuration. For example, all L-isomersof α-amino acid have identical chiral configuration. Thus, in thegeneral α-amino acid structure, R^(a)R^(b)C(NH₂)COOH, wherein R^(a) ishydrogen, hydrocarbyl, substituted hydrocarbyl or heterocyclo; and R^(b)is hydrogen, the —COOH, —NH2, R^(a), and R^(b) constituents of L-isomersof α-amino acid have the same spatial arrangement around the chiralcarbon. Similarly, the two or more L-enantiomers of a specific α-hydroxycarboxylic acid, such as two molecules of L-HMB, will have identicalconfiguration to each other.

[0187] The term “enantioselective” refers to the selection of a specificenantiomer of an enantiomeric mixture and interactions with saidenantiomer. For example, an enantioselective enzyme, such as papain,selectively catalyzes the linkage of L-methionine ethyl esters to forman oligomer of L-methionine residues.

EXAMPLES

[0188] The following Examples set forth one approach that may be used tocarry out the process of the present invention. Accordingly, theseexamples should not be interpreted in a limiting sense.

Example 1

[0189] Synthesis of Methionine Oligomers and HMB-Methionine Co-oligomers

[0190] This example demonstrates the enzymatic synthesis of oligomerscomprising methionine and co-oligomers comprising HMB-methionine, aswell as their characterization using reverse-phase HPLC and matrixassisted laser desorption ionization-time of flight mass spectroscopy(MALDI-TOF MS) analysis.

[0191] Co-oligomerization

[0192] In a first synthesis, the experiment was generally conducted inaccordance with reaction conditions for the papain-catalyzedoligomerization of methionine analogs as described by Arai et al. inAgric. Biol. Chem., 43(5), 1069-1074 (1979). The oligomerizationcomprised forming a reaction mixture at a temperature of 37° C.consisting of nanopure filtered water (10 mL) containing HMB ethyl ester(0.7 M) and methionine ethyl ester (0.7 M) along with L-cysteine (0.1M), EDTA (10 mM), sodium citrate (1M) and 1% papain (by weight of themonomer) at a pH of 5.5. The mixture was allowed to incubate for 24hours. After 10 minutes, aliquots were removed at regular intervals tomonitor the degree of oligomerization and co-oligomerization and thedisappearance of the substrate.

[0193] In a second synthesis, the experiment was conducted in accordancewith reaction conditions for the papain-catalyzed oligomerization ofmethionine analogs as described by Jost et al. in Helv. Chim. Acta, 63(1980) 375-384 (1980). The oligomerization comprised forming a reactionmixture consisting of L-methionine ethyl ester (5 g) and HMB ethyl ester(5 g) dissolved in nanopure water (50 mL) containing sodium bicarbonatebuffer (0.1 mole) and L-cysteine (4 mmole). The pH was adjusted to 9 andthe solution was made up to 100 mL and incubated for 24 hours at 37° C.after adding papain (2 g).

[0194] Analysis of Oligomers

[0195] In all cases, aliquots were removed from the oligomerizationreaction mixtures and heated to 80° C. for 10 minutes to denature theenzyme. The mixture was centrifuged and the supernatant was analyzed ona reverse-phase HPLC to monitor the synthesis of methionine oligomers oforder 3 or less along with the disappearance of the substrate. Attemptsat resolving the higher order oligomers with RPLC and gel permeationliquid chromatography (GPC) were unsatisfactory especially for oligomerswith 4-10 monomer residues. For example, the experiments revealed thatunderivatized oligomers could not be eluted from C-18 or C-8 columnswith the common mobile phases due to poor solubility of oligomers inthese mobile phases. The oligomers were soluble in dimethyl sulfoxide(DMSO) and tetrahydrofliran (THF) a common mobile phase in GPC forseparations. However, oligomers with less than ten residues could not beresolved from the solvent in GPC separations. A persulfonation procedurewas therefore adopted. Persulfonation of oligomers enhanced the polarityof the oligomers to a point that these could be separated on a C-18column with a moderately polar mobile phase (M. Spindler, R. Stadler andH. J. Tanner, J. Agri. Food Chem., 32(6) (1984)1366-1371).

[0196] Persulfonation of Oligomers

[0197] The problem of analysis of higher order oligomers was addressedby the oxidation of the methionine and the HMB to their relativelyhydrophilic sulfones with performic acid. The mixture was washedthoroughly until no traces of the monomers and the salts were leftbehind. The mixture was then freeze dried and a part of it was subjectedto persulfonation with a method which was adapted from a procedureoutlined by Spidler and coworkers. The procedure involved oxidation ofall sulfide moieties in the oligomers with performic acid. The performicacid for the purpose was prepared by oxidation of formic acid (HCOOH)with hydrogen peroxide (H₂O₂). A solution of 30% H₂O₂ (0.5 mL) was mixedwith 88% HCOOH (4.5 mL) and phenol (25 mg). The mixture was allowed tostand for 30 minutes at room temperature. After 30 minutes, the mixturewas cooled to 0° C. for 15 minutes in an ice bath. The finely dividedoligomer powder (10 mg) was then contacted with the performic acidmixture in the ice bath. After stirring for 15 minutes, theoligomer—performic acid mixture was placed in a refrigerator overnight.The excess performic acid was reduced with 48% hydrobromic acid (0.7mL). The residual bromine and formic acid were removed with a rotaryevaporator at 50-60° C.

[0198] Liquid Chromatography

[0199] The oligomer sulfone residues in the rotary evaporator roundbottom flasks were dissolved in a 40:60 acetonitrile/water mixture (5mL) and filtered through a membrane filter. A 10 μL aliquot of thesolution was injected into a HPLC. The separation of persulfonatedoligomers was achieved with a C-18 column using a phosphatebuffer—acetonitrile mobile phase. A linear gradient was used tofacilitate separations. In this gradient the mobile phase compositionwas changed from 100% eluant A (phosphate buffer, pH 6.5) to 60% A and40% B (20% Acetonitrile) in 20 minutes. The mobile phase flow rate wasmaintained at 1 mL min⁻¹. The separated oligomers were detected with aUV/VIS diode array detector.

[0200] TOF Experiments

[0201] Aliquots of purified oligomers dissolved in DMSO were introducedinto the mass spectrometer along with a thioglycerol matrix. The massspectrometer operating parameters were: Accelerating Potential +20K VGrid Voltage 80% Low Mass Gate 191.0 Flight tube pressure 3.3 e⁻⁷ torr

[0202] MALDI-TOF Analysis

[0203] The MALDI-TOF spectra of methionine (Met) oligomers are shown inFIG. 1. The spectra contain distinct ions which are separated by mass131. This mass (131) represents the repeating Met moiety (C₅H₉NOS),since the masses of the N and C terminal methionine residues are 132 and148 respectively. Therefore, a methionine hexamer (Met)⁶,(^(N)Met-(Met)₄-Met^(C))+H⁺ should appear at m/z 805 and (Met)⁷ shouldappear at m/z 936. However, the m/z values of the dominant ions did notcorrespond to this series, instead, one set of dominant ions appeared atm/z 826, 957, 1088, 1219, 1350 and 1481. These ions most likelycorrespond to ((Met)^(n)+Na⁺), where n is an integer between 6 and 11.The second group of ions appeared at m/z 724, 855, 986, 1117, 1248 and1379. These ions most likely correspond to the series(^(N)Met-(Met)-Met-O—C₂H₅)+Na⁺. A third set of ions appeared at m/z 739,870, 1001 and 1134, these ions most likely correspond to^(N)Met-(Met)_(n)-Met-O—C₂H₅+K⁺. A fourth set of unidentified ions seemto be present at regular intervals in the clusters and which might beassigned the mass values, 842, 973, 1104, 1235 and 1366 corresponding tothe series ^(N)Met-(Met)_(n)-Metc)+K⁺. In all these cases “n” variedbetween 6 to 11.

[0204] The spectra of HMB—Met co-oligomers is shown in FIG. 2. In thisspectra, ions corresponding to the series(^(N)Met-(Met)_(n)-Met^(C))+Na⁺, (^(N)Met-(Met)_(n)-Met-O—C₂H₅)+Na⁺,^(N)Met-(Met)_(n)-Met^(C)+K⁺, and ^(N)Met-(Met)_(n)-Met-O—C₂H₅K⁺ werereadily observed. However, the ions, which should correspond to(HMB-(Met)_(n)-Met^(C))+H⁺ or +Na⁺ m/z 806, 937 and 1118; 827, 958 and1089 were not observed in the spectra. The apparent absence of theseions, however, does not necessarily mean the absence of HMB-(Met)^(n)co-oligomers in the product mixture. The absence of the ions can beattributed to two factors. The first relates to the low resolving powerof the MALDI-TOF MS, which would prevent the resolution of theH⁺HMB-(Met)^(n)-Met^(C) ions at m/z 806, 937, 1118 from theH^(+N)Met-(Met)^(n)-Met^(C) ions at m/z 805, 936, 1117. The second andmore probable cause is the low intensity of the H⁺HMB-(Met)^(n)-Met^(C)ion due to the absence of a good protonation site in HMB-(Met)^(n)co-oligomers.

[0205] HPLC Separations of Oligomers Sulfones

[0206] The chromatographic separations of poly-methionine sulfones areshown in FIG. 3. A number of well-resolved peaks can be readilyobserved. Of these, nine did not appear in the reagent blank and mostlikely represent the poly-methionine sulfones. This chromatographicseparation is nearly identical to the chromatographic separationsreported by Kasai et al. (T. Kasai, T. Tanaka, and S. Kiriyama, Biosci.Biotech. Biochem.,56(11) (1992) 1884-1885). However, because of adifference in the separation column or variations in the eluantcomposition, the retention times reported by Kasai et al. for mostoligomers were approximately 0.5-0.6 minutes longer than retention timesobserved by the present inventors.

[0207] The chromatographic separation of HMB-poly-methionine sulfones isshown in FIG. 4. This chromatogram contained a number of peaks, whichwere not present in the poly methionine sulfone chromatogram. Thisindicates that HMB is incorporated in the (Met)^(n) oligomer. Theincorporation most likely occurs at the N-terminal end. The resultingHMB-(Met)^(n) co-oligomers, with the terminal hydroxyl, should be lesspolar than the corresponding (Met)^(n) oligomers with the terminal aminemoiety. Therefore, the HMB containing co-oligomers should elute laterthan the corresponding Met oligomers and this appears to be the case.The elution times for methionine sulfones and HMB methionine sulfonesare given in Table 1. The chromatographic separations of methionineoligomer (sulfones) obtained after different incubation periods indicatethat the relative abundance of methionine oligomers is dependent on theincubation period. The abundance of longer chain co-oligomers was higherin co-oligomers obtained after 24 hours incubation (FIG. 5) relative tothe co-oligomers obtained after 10 minutes incubation (FIG. 4). It canbe readily observed that the concentrations of longer chain co-oligomersincreased with an increase in the incubation period. Chromatographicresults also indicate that presence of HMB may affect the relativedistribution of methionine oligomers. These results are significant inlight of the reports in the literature, which suggest that the uptake ofmethionine oligomers is dependent on the size of the oligomers. TABLE 1Elution Times of Met Oligomer and HMB-Met Co-Oligomer Sulfones ElutionTime (mins) Elution Time (mins) Oligomer Present Study Kasai et al.(Met)₄ 10.0 NR HMB-(Met)₃ 11.8 NA (Met)₅ 13.4 14.0 HMB-(Met)₄ 15.1 NA(Met)₆ 16.8 17.8 HMB-(Met)₅ 18.5 NA (Met)₇ 20.1 21.0 HMB-(Met)₆ 21.8 NA(Met)₈ 23.3 24.0 HMB-(Met)₇ 24.9 NA (Met)₉ 26.5 26.9 HMB-(Met)₈ 28.6 NA(Met)₁₀ 31.1 29.5 HMB-(Met)₉ 34.2 NA

Example 2

[0208] Oligomerization and Co-Oligomerization of Lysine and HMB

[0209] This example demonstrates four alternative procedures for theenzymatic synthesis of oligomers comprising lysine and co-oligomerscomprising HMB-lysine. The experiment was designed to compare threenovel synthesis procedures to that of Puigserver et. al.¹ who reported aprocedure for papain catalyzed polymerization of lysine.

[0210] In general, protease-catalyzed synthesis of water insoluble aminoacid oligomers in aqueous media is driven by precipitation. Thesynthesis of water soluble oligomers of amino acids, such as lysine canbe controlled only in mixed phase systems where the equilibria isshifted in favor of the synthesis of polypeptides due to enhancedpartitioning of peptide in the organic phase. Puigserver et. al.reported a procedure for papain catalyzed polymerization of lysine whichinvolved the binding of papain to modified PEG (MW 2000 or 5000). Thebound enzyme was then used to synthesize poly lysine in a two phasereaction mixture. The following summarizes the experiment to usePuigserver's method to explore the feasibility of the co-oligomerizationof lysine and HMB in comparison to three novel synthesis methods.

[0211] PEG bound Papain system (Puigserver's Method): 10 mM of substratewas added to 98 mL of toluene along with 0.8 mL of Diisopropyl aminoethyl and 0.2 mL of mercaptoethanol, followed by 17 mM of PEG₂₀₀₀modified Papain. The mixture was allowed to incubate for 24 hours,before being evaporated and redissolved in deionized water and analyzedon a ion-pair liquid chromatography column.

[0212] Two Phase Toluene:Water System: This solvent system was evaluatedwith varied phase ratios, two of which are described below:

[0213] a) 10 mM of substrate was added to 98 mL of toluene along with0.8 mL of Diisopropyl amino ethyl and 0.2 mL of mercaptoethanol,followed by 1 mL of aqueous papain suspension. The mixture was allowedto incubate for 24 hours, before being evaporated and redissolved in DIwater and analyzed on a ion-pair liquid chromatography column.

[0214] b) 100 mM of substrate was added to 8.9 mL of toluene along with0.08 mL of Diisopropyl amino ethyl and 0.02 mL of mercaptoethanol. Thiswas followed by 1 mL of aqueous papain suspension, which resulted in atwo phase system. The mixture was allowed to incubate for 24 hours,before being evaporated and redissolved in DI water and analyzed on anion-pair liquid chromatography column.

[0215] Reverse Micellar System: 10 mM of substrate was dissolved in 98mL of a reverse micellar solution containing 150 mM of AOT (3.33 g), 0.8mL of diisopropyl amino ethyl and 0.2 mL of mercaptoethanol inisooctane. 1 mL of aqueous papain solution was added to the mixture andallowed to incubate for 24 hours, at the end of which the mixture washeated to denature the enzyme and the oligomeric and co-oligomericproducts extracted with 1 M NaCl solution. The solution was lateranalyzed on a ion-pair liquid chromatography column.

[0216] Three Phase DFP:Octane:Water System: To a two phase systemcomprising of 4.45 mL of DFP and 4.45 mL of octane was added 100 mM ofsubstrate along with 0.08 mL of Diisopropyl amino ethyl and 0.02 mL ofmercaptoethanol. The addition of 1 mL of aqueous papain suspension whichis insoluble in either of the phases converts this system to a threephase system. The mixture was allowed to incubate for 24 hours, beforebeing evaporated and redissolved in DI water and analyzed on a ion-pairliquid chromatography column

[0217] Results: The yield and the degree of oligomerization andco-oligomerization were determined with ion-pair liquid chromatographyand MALDI-TOF mass spectrometry. These results appear in FIGS. 6 to 13.In general, Puigserver's method was found to be cumbersome and did notyield any discernable HMB-lysine co-oligomers. Reaction conditions andresults are further summarized in Table 2. TABLE 2 Procedure for thevarious methods used to synthesize lysine oligomers and HMB-lysineco-oligomers Lysine Oligomerization HMB-lysine Co-oligomerizationComponents Pgsr^(f) 2-f^(g) RM^(h) 3-f^(i) RV 2-f^(j) Pgsr^(f) 2-f^(g)RM^(h) 3-f^(i) RV 2-f^(j) LysEE.2HCl (mM) 10 10 10 100 100 5 10 10 50 50HMB analog mM 5 10 10 50 50 i-Pr₂NH₂Et (% v/v)^(a) 0.8 0.8 0.8 0.8 0.80.8 0.8 0.8 0.8 0.8 SCH₂CH₂OH (% v/v)^(b) 0.2 0.2 0.2 0.2 0.2 0.2 0.20.2 0.2 0.2 Papain (% v/v)^(c) 1 1 10 10 1 1 10 10 Toluene (% v/v) 98 9889 98 98 0 89 Isooctane (% v/v) 98 98 DFP (% v/v)^(d) 44.5 44.5 Octane(% v/v) 44.5 44.5 AOT (mM)^(e) 150 150 PEG-Papain (mM) 17 17 Yields (%)0 17 22 95 90 0 17 22 95 90

Example 3

[0218] HMB-methionine and HMB-lysine co-polymers were synthesizedenzymatically through a papain-catalyzed reaction along withpoly-methionine and poly-lysine (as controls) as described in Examples 1and 2. The biological release of the amino acids from the oligomers wasexamined using several digestive enzymes including pepsin, trypsin,chymotrypsin, intestinal peptidase and carboxypeptidase. The oligomerswere dissolved at 10 mg/mL in 0.15 HCl (pH 2.5) or 50 mM KPO₄ (pH 7.5).Samples (0.5 mL) were incubated with 10 units of each enzyme for 2 hoursat 37° C. The extent of digestion was quantified by measurement of newlyreleased amino groups and their reaction with o-Phthalaldehyde (OPA) and2,4,6-trinitrobenzene sulfonic acid (TNBSA). Acid hydrolysis wasprepared by complete hydrolysis of 10 mg/mL polymers in 6 M HCl for 24hours at 110° C. Results are summarized below in Table 3.

[0219] Results show that HMB-methionine and HMB-lysine can be hydrolyzedby strong acid and heat. HMB-met is digested only 3.5% by pepsin and notat all by the other proteases. Poly-lysine can be digested by intestinalpeptidase (20% in 2 hours at 37° C.) but not by other proteases.HMB-lysine is not digested by any of the proteases tested. Inconclusion, these data suggest the lack of enzymatic digestion ofHMB-met and HMB-lysine co-oligomer was caused by a structural differenceinstead of solubility of the co-oligomers. TABLE 3 ENZYMATIC DIGESTIONOF AMINO ACID POLYMERS Poly-Lys HMB- HMB- Poly-Lys Enzyme (˜8mer) metPoly-met Lys (˜4mer) pepsin 0 0.030 0.062 0 0 (3%) (15%) trypsin 0.008 00 0 0 (15%) chymotrypsin 0 0 0 0 0 intestinal 0.013 0 0.052 0 (TNBSA,peptidase (25%) (13%) ˜20%) carboxy- 0 0 0 0 0 peptidase A acid 0.0520.868 0.406 0.063 0.093 hydrolysis (-initial value)

Example 4

[0220] Characterization of Methionine Oligomers and HMB-MethionineCo-oligomers

[0221] Met oligomers and HMB-Met co-oligomers produced through papainmediated enzymatic reactions at pH 5.5 and pH 9.0 according to theprocedure described in Example 1 were subjected to persulfonation.Persulfone derivatives were separated with the reverse phase liquidchromatography (RPLC). The separated oligomers and co-oligomers weremonitored with a UV absorption spectrophotometeric detector and anelectrospray ionization interface (ESI) mass spectrometer. Theabsorption wavelength was set at 210 nm. The mass spectrometer wasoperated in positive and negative ion modes. The outputs of the UVabsorption detector and the positive ion ESI-MS are shown in FIGS. 14and 15.

[0222] The chromatogram of (Met)n persulfones obtained with the UVdetector and the positive ion total ion chromatogram (TIC) were similarto the chromatograms obtained from earlier with a earlier experimentsusing a RPLC-Diode Array Detector (DAD) system. These results hadindicated the formation of Met homo-oligopeptides and HMB-Metco-oligomers. The results were supported by data obtained from thematrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)experiments.

[0223] The experiments with ESI-MS in the positive ion mode confirmedthe formation of methionine oligomers. The ESI-MS spectrum forindividual LC peaks provides conclusive evidence for the formation of(Met) to (Met) oligomers. The mass difference between in the molecularmasses of successive oligomers was found to be 163, which corresponds tothe sulfonated methionine residue.

[0224] Spectra corresponding to different peaks are shown in FIGS.16-20. The general formula for the separated oligomers is:

[0225] The positive ion TIC of HMB-(Met)n co-oligomers obtained withESI-MS did not contain extra peaks observed in the LC-UV chromatogram.The spectra of individual peaks in the HMB-Met co-oligomers did notprovide any evidence for HMB-(Met)n co-oligomers formation. Theseresults were not unexpected, the lack of pseudo-molecular positive ionsin the MALDI-TOF spectra of HMB-(Met)n in the earlier experiments hadled us to the conclusion that HMB is attached at the N-terminal end ofthe polymethionine chain. The lack of protonated ions in the HMB-Metco-oligomers is the result the weak proton affinity of the terminalhydroxyl group.

[0226] The confirmation of the HMB-(Met)n was obtained by monitoringnegative ions formed through electron attachment to the (Met)n andHMB-(Met)n chains. The TIC of HMB-(Met)n in this case contained extracomponents (peaks) which corresponded to the extra peaks observed in theLC-UV chromatograms FIGS. 21 and 22.

[0227] A few representative spectra for the HMB-(Met)_(n) peaks areshown in FIGS. 23-25. As expected, the molecular ions for HMB-(Met)nappear at one mass unit higher than the corresponding (Met)n ions. Inaddition, the retention times of HMB-(Met)n peaks are longer than thecorresponding (Met)n peaks. This is to be expected since the terminalamine group of the (Met)n imparts higher polarity to methionineoligomers than the terminal hydroxyl to the HMB-(Met)n co-oligomers.

[0228] The presence of sulfonated methionine residue in both (Met)noligomers and the HMB-(Met)n co-oligomers chains is again revealed bymass difference of 163 amu between the molecular masses of the separatedchromatographic peaks. The mass difference corresponds to the mass ofthe methionine sulfone residue.

[0229] Both the positive ion and negative ion LC-ESIMS data show thatthe predominant (Met)n are composed of four to ten methionine residues.Likewise, the negative ion LC-ESI data shows that the predominantHMB-(Met)n co-oligomers contain one HMB residue and four to ninemethionine residues. The relative distribution of (Met)n oligomers andHMB-(Met)n co-oligomers is presented in FIGS. 26 and 27.

Example 5

[0230] Synthesis of HMB-(Met)n Co-oligomers with HMB-methyl Ester andMet-ethyl Ester ESI-MS Results

[0231] Further experiments were conducted to confirm that HMB isattached at the N-terminal of the oligomers chain. In one suchexperiment methyl ester of HMB and ethyl ester of methionine wereprepared. Equivimolar amounts of mixed esters were subjected to papainmediated oligomerization and co-oligomerization at pH 5.5 with theprocedure outlined in Example 1. The product was washed with water untilit was free of monomers. The product was then freeze-dried, dissolved inDMSO and introduced in the MS through an ESI interface. The positive andnegative ion spectra obtained for the mixed co-oligomers are shown inFIGS. 28A and 28B respectively.

[0232] The positive ion spectra (FIG. 28A) of shows two series of ionsthat are 28 mass units apart. One series containing ions m/z 674, 805,936, 1067, 1198 and 1229 correspond to ((Met)_(n)+H⁺) ions. The otherseries of ions which occur at m/z 702, 833, 964, 1095, 1226 and 1357correspond to ((Met)n-OET)+H⁺ ions. Ions in both series are 131 massunits or one methionine residue (C₅H₉NOS) apart. In both series, thevalue of n lies between 4-10.

[0233] Incorporation of HMB at the C-terminal end of the polymethioninechain should have resulted in a series of ions corresponding to((Met)n-HMB-OCH₃)+H⁺ ions. Such ions, if formed, would appear at m/z688, 819, 958, 1081, 1212 and 1343. However, none of the peaks of thisseries were observed in the spectra. Similar results were obtained inthe case of the negative ions (FIG. 28B). The absence of the methylgroup provides indirect evidence that HMB is incorporated at theN-terminal end. It should be pointed out that dominant ions obtained innegative ion mode were adduct ions and contained a dimethyl sulfonemoiety.

Example 6

[0234] Sonic Spray—MS-MS Results

[0235] The polymethionine and HMB-polymethionine prepared from Met-ethylester and HMB-ethyl ester were also subjected to MS-MS experiments. Thefreeze-dried precipitates were dissolved in DMSO (2 μg/μl) and thesolution was introduced into the mass spectrometer with the sonic sprayinterface at the rate of 1 mL/hr. The solution was mixed with 1:1acetonitrile:water mixture containing 0.1% acetic acid. The total fluidvolume entering the SSI-MS was maintained at 0.2 mL/min. The parent ionand spectra obtained with the system are shown in FIG. 29.

[0236] Ions of (Met)n-OEt+H⁺ corresponding to methionine hexamer,heptamer, octamer and nanomer were observed at m/z 833, 964, 1095 and1225 amu respectively. For MS-MS experiment, the ion at m/z 833 wasexcited with an auxiliary Vrf and subjected to collision induceddissociation. The daughter spectrum of (Met)₆-EE is shown in FIG. 30.The prominent fragment ion was observed at m/z 657, this ion resultsfrom the cleavage of the amide bond resulting in the loss of Met-OEt(C₇H₁₄NO₂S) moiety from the C-terminal end. Similar results wereobtained with molecular ions resulting from HMB-(Met)_(n) ⁻. Daughterions resulting from the loss of HMB-O Methyl (C₆H₁₂NO₂S) moiety were notobserved, indicating the absence of HMB-O Methyl at the C-terminal end.

Example 7

[0237] Papain Catalyzed Synthesis of HMB-Tyrosine Co-oligomers

[0238] Synthesis of tyrosine oligomers and HMB-tyrosine co-oligomers wasinitiated with tyrosine ethyl ester (Tyr-OMe) and HMB ethyl ester as themonomer substrate. The overall synthesis and purification approach wassimilar to the one used for methionine and HMB-methionine described inExample 1.

[0239] Tyr-OMe (equal amounts (wt %) in the case of HMB-OEt and Tyr-OMe)were dissolved in 9.5 mL of 1M citrate buffer. EDTA and L-Cysteine wereadded. The reaction mixture was adjusted to a pH of 5.5 and 0.5 mL ofpapain suspension was added to the mixture. The reaction mixture wasincubated in a shaker for 24 hours. The enzyme was then denatured byheating the reaction mixture to 80° C. for 10 minutes. The reactionmixture was cooled to room temperature.

[0240] The reaction mixture was filtered to collect the precipitate. Theoligomer and co-oligomers in the precipitate were dissolved in DMSO toseparate them from the monomers which are relatively insoluble in thesolvent. The separated solvent was then evaporated to re-precipitate theoligomer and co-oligomers, which were then washed with water andfreeze-dried.

[0241] The recipe for the tyrosine oligomers and HMB-tyrosineco-oligomers experiment are provided in Table 4. TABLE 4 ReactionMixtures Used for Tyrosine Oligomerization and HMB-TyrosineCo-Oligomerization Components MW Moles Wt AA-ester 3 g L-Cys.HCl.H₂O175.6 100 mM 0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g Na Citrate 294.11 M 2.941 Papain 21428D 7*10⁻⁵ M 15 mg Volume 10 mL pH 5.5

[0242] The reaction achieved a reaction rate similar to that observedwith methionine in Example 1. Approximate oligomer yield was 70-80%. Thefreeze-dried oligomer precipitates were solubilized in DMSO. Thesolution concentration was brought to approximately 2 μg/μl. Thesolution was mixed with 1:1 acetonitrile:water mixture containing 0.1%acetic acid. The total fluid volume entering the ESI-MS was maintainedat 0.2 mL min.⁻¹ The positive mass spectrum of the tyrosine oligomers isshown in FIG. 31.

[0243] Two sets of prominent ions appeared in the Tyr oligomers spectra.One series of ions appeared at m/z 834, 997, 1160 and 1323, while theother series of ions were found at m/z 862, 1025, 1188 and 1351. Theions in the first series represent (Tyr)n+H⁺, while the ions in thesecond series represent (Tyr)n-OEt+H⁺. The ions in the two series are 28amu (C₂H₄) apart indicating the presence of O-Et at the C-terminal endin one series. Ions within the two series are separated by 163 amu,corresponding to the repeating unit of the Tyr residue (C₉H₉NO₂). Thus,the protonated ion at 862 most probably represents the tyrosineoligomers with five residues and an ethyl ester attached to theC-terminal end. Similarly, the ion at m/z 1025 most likely results fromthe (Tyr)⁶⁻OEt+H⁺. The ion at m/z 997 results from (Tyr)₆+H⁺. Thepresence of oligomers with 5 to 8 Tyr residues is clearly evident,furthermore, the (Tyr)₆ was found to be the most prominent oligomer.

[0244] The positive ion mass spectrum of HMB-tyrosine co-oligomer isshown in FIG. 32A. The dominant ions in this spectrum were the same ionsobserved in the positive ion spectrum of polytyrosines, FIG. 31A.However, additional ions appeared at m/z 831, 994 and 1157. These ionsappear at a mass difference of 133, suggesting the presence of a HMBresidue in the co-oligomer. The peak at m/z 831 most probably representsthe co-oligomer with one HMB residue and 4 tyrosine residues with theethyl ester moiety (HMB-(Tyr)₄OEt+H⁺). Similarly, the residues at m/z994 and 1157 represent co-oligomers with one HMB residue and 6 and 7tyrosine residues respectively. The weak intensity of these ions in partrelate to lower proton affinity of the hydroxyl group.

Example 8

[0245] Papain Catalyzed Synthesis of HMB-Leucine Co-oligomers

[0246] The papain-catalyzed synthesis of leucine and HMB co-oligomerswas performed. Synthesis of leucine oligomers and HMB-leucineco-oligomers was initiated with leucine ethyl ester and HMB ethyl esteras the substrates. The overall synthesis and purification approach wassimilar to the one used in the case of methionine and HMB-methionine.Reaction rates similar to those obtained with methionine and tyrosinewere achieved. Approximate oligomer yield was 58%. The freeze-driedoligomer precipitates were solubilized in DMSO. The solutionconcentration was brought to approximately 2 μg/μl The solution wasmixed with 1:1 acetonitrile:water mixture containing 0.1% acetic acid.The total fluid volume entering the ESI-MS was maintained at 0.2 mLmin.⁻¹ The positive mass spectrum of the leucine oligomers is shown inFIG. 33A.

[0247] Four sets of ions appeared in the positive ion spectra of(Leu)_(n). One set of ions corresponding to (Leu)_(n)+H⁺ appeared at m/z698, 811 and 924. The other set of ions appeared at m/z 720, 833 and 947and correspond to (Leu)_(n)+Na⁺. Another set of ions appeared at m/z747, 860 and 973 which correspond to (Leu)₆-OEt+Na⁺, (Leu)₇-OEt+Na⁺ and(Leu)₈-OEt+Na⁺. However, the two prominent ions in the spectra appear tobe (Leu)₆-OEtNa+Na⁺ and (Leu)₇-OEtNa+Na⁺. These results clearly showthat the dominant oligomers are (Leu)₅, (Leu)₆, (Leu)₇ and (Leu)₈. Themass difference of 28 amu (C₂H₄) indicates the presence of O-Et at theC-terminal. Ions within the two series are separated by 113 amu,corresponding to the repeating unit of the Leu residue (C₆H₁₁NO). Thus,the doubly sodiated (Na₂) ion at m/z 770 and 883 most probablyrepresents leucine oligomers with six and seven residues and a ethylester attached to the C-terminal end.

[0248] The negative ion mass spectrum of the leucine oligomers is shownin FIG. 33B. The overall appearance of the spectra is similar to that ofthe positive ion spectra. The two dominant ion in this spectracorrespond to (Leu)₆-OEt+Na and (Leu)₇-OEt+Na.

[0249] The positive and the negative ion spectra of HMB-Leu co-oligomersare shown in FIG. 34. As expected, the positive spectra contained all ofthe dominant ions observed in the positive ion ESI-MS spectra of(Leu)_(n). However, three additional strong ions at m/z 740, 853 and 866were also found. These masses correspond to sodiated co-oligomersHMB-(Leu)₅+Na⁺, HMB-(Leu)₆+Na⁺ and HMB-(Leu)₇+Na⁺ respectively. Thus,formation of co-oligomers with one HMB residue with five to sevenleucine residues is clearly evident.

Example 9

[0250] Papain Catalyzed Synthesis of HMB-Phenylalanine Co-oligomers

[0251] Papain catalyzed synthesis of phenylalanine and HMB co-oligomerswas also conducted. Synthesis of phenylalanine oligomers and HMB-phenylalanine co-oligomers was initiated with phenylalanine ethyl esterand HMB ethyl ester as the substrates. The overall synthesis andpurification approach was similar to the one used in the case ofmethionine and HMB-methionine in Example 1. The oligomerization reactiondid not proceed when phenylalanine was the only substrate present in thereaction mixture. The reaction did proceed when HMB-ethyl ester wasadded to the reaction mixture. Reaction rates similar to those withmethionine and tyrosine were achieved. Approximate oligomer yield was90%. The freeze-dried oligomer precipitates were solubilized in DMSO.The solution concentration was brought to approximately 2 μg/μl. Thesolution was mixed with 1:1 acetonitrile:water mixture containing 0.1%acetic acid. The total fluid volume entering the ESI-MS was maintainedat 0.2 mL min.⁻¹ As stated earlier, phenylalanine homo-oligomers werenot formed.

[0252] The ESI-MS results of HMB-PheHMB co-oligomerization reaction aregiven in FIG. 35. The positive ion spectra of HMB-Phe co-oligomers aredepicted in FIG. 35A, while the negative ion spectra are depicted inFIG. 35B. The positive spectra of the co-oligomers yielded three ionpeaks at m/z 790, 937 and 1084. The mass difference between these ionsis 147 or a difference of one phenylalanine residue (C₆H₆NO=147). Them/z values of the ions most likely correspond to HMB-(Phe)₄-OEt+Na⁺,HMB-(Phe)₅-OEt+Na⁺ and HMB-(Phe)₆-OEt+Na⁺. Thus, formation ofco-oligomers with one HMB residue and four to six Phe residues isclearly evident.

Example 10

[0253] Optimization of (Lys)n Oligomers and HMB-(Lys)n Co-oligomersSynthesis

[0254] Experiments were conducted to optimize the reaction conditionsfor papain catalyzed synthesis of lysine oligomers and lysineco-oligomers with HMB. Reactions were carried out in two systems. Thefirst system consisted of an aqueous phase and an immiscible organicphase, while the second system comprised a three-phase system consistingof an aqueous phase sandwiched between two mutually immiscible organicphases.

[0255] A. Two Phase Reaction System

[0256] The two-phase reaction system consisted of a small amount ofpolar phase and a larger amount of an immiscible non-polar phase. Thepolar phase comprised water, isopropyl amino ethyl and mercaptoethanol.This phase also contained the amino acid ester substrate and papain.During optimization, parameters such as the volume ratio of the aqueousand the non-aqueous phase, composition of additives, concentrations ofthe additives, concentrations of the substrates, and the concentrationof the enzyme were varied. The effect of these parameters on the degreeof oligomerization and yield were monitored. The results of theexperiments are summarized below:

[0257] A.1 Aqueous: Organic Phase Ratio

[0258] To optimize the volume ratio of the aqueous and organic phases(toluene), the oligomer yields and degree of oligomerization weremonitored over phase ratios ranging from 1:9-1:99, the reaction wasallowed to proceed for 24 hours. Oligomer were recovered from theaqueous phase and analyzed. Results of these experiments are shown inFIG. 36.

[0259] The results indicate that the yields dropped at ratios below 19and the higher ratios did not lead to an appreciable change in totalyield. The results also showed that while the total yield did not changeat higher organic solvent volumes, the degree of oligomerization wasaffected. Higher toluene volume led to a decrease in the degree ofoligomerization. The length of oligomers chains at phase ratio 1:19extended up to nine lysine residues (Lys)₉, whereas at phase ratio 1:39,the largest oligomers contained only six lysine residues (Lys)₆, FIG.37. In light of these results and to conserving organic solvent, allsubsequent experiments were carried out at phase ratio 1:19.

[0260] A.2 Optimization of Additive Concentration

[0261] The effect of the concentration of additives (mercaptoethanol andisopropyl ethyl amine) on yield and degree of oligomerization was alsoexamined. These additives act as antioxidants and prevent oxidation ofcysteine moiety in the enzyme. The reaction was carried out for 24hours. Oligomers were recovered from the aqueous phase and analyzed.Results showed that concentration of additives had a marked effect onthe total yield and the degree of oligomerization. The total yieldincreased with an increase in additive concentration from 0.1-2%.However, a pronounced decrease in total oligomers yield was observedwhen the additive concentration was increased above 5%. A 2% additiveconcentration was found to the optimum under conditions used in theseexperiments. The total oligomers yield at this additive concentrationwas approximately 87%, FIG. 38.

[0262] The degree of oligomerization was found to increase with anincrease in additive concentration up to 2%, still higher concentrationsdid not lead an appreciable change in the oligomers distribution asshown in FIG. 39.

[0263] A.3 Optimization of Substrate Concentration

[0264] A series of experiments were carried to optimize the substrate(lysine ethyl ester) concentration at a fixed enzyme activity. Theconcentration of substrate was varied five folds, from 500 mM to 2,500mM, while the enzyme concentration was held constant at 1.21 mM.Oligomerization reactions were allowed to proceed for 24 hours afterwhich the enzyme was deactivated and oligomers recovered from theaqueous phase and analyzed. A plot of the percent oligomers yield (totaloligomers mass/total substrate mass×100) vs the substrate mass is shownin FIG. 40.

[0265] The results show that the highest conversion efficiency wasachieved at a substrate concentration of 1000 mM. A noticeable drop inconversion efficiency above this concentration was clearly evident. Thedegree of oligomerization was also affected by the substrateconcentration. In general, higher concentration led to the formation ofoligomers with higher lysine residues (e.g., the most abundant oligomersat 500 mM lysine ethyl ester was (Lys)₄ and the yield of higher homologswas quite low). The most abundant oligomer was (Lys)₅. In addition,concentrations of higher homologs (Lys)₆, (Lys)₇ and (Lys)₈ werenoticeably higher, FIG. 41.

[0266] A.4 Optimization of Incubation Period

[0267] Another set of experiments was carried out to determine anoptimum incubation period for oligomerization of lysine. The reactionwere conducted with 1:19 phase ratio, 1 M substrate concentration and 1%additive concentration. The reaction was allowed to continue for timeperiods ranging between 30 minutes to 24 hours. After each time period,the reaction was brought to halt by deactivating the enzyme. Theoligomers were recovered from the aqueous phase and analyzed. The totaloligomers yield obtained at various time periods is shown in FIG. 42.

[0268] The graph shows that the reaction is nearly complete within thefirst four hours and only a marginal increase in yield is obtained atlonger incubation periods. Analyses of oligomers obtained afterdifferent time periods showed that the time periods shorter than 4 hoursyield an even distribution of oligomers from (Lys)₂ to (Lyn)₈, while thelonger periods yield higher concentrations of (Lyn)₄ to (Lys)₆oligomers, FIG. 43.

[0269] B. Three Phase System

[0270] The three-phase system consisted of an aqueous phase presentbetween two immiscible non-aqueous phases, one lighter than the aqueousphase and the other heavier than the aqueous phase. The heavier phasecomprised decafluoropentane and the lighter phase comprised n-octane.Isopropyl ethyl amine and mercaptoethanol additives were added to theaqueous phase along with the lysine ethyl ester (substrate) and papain(enzyme). The effects of parameters such as the relative volumes ofaqueous to non-aqueous phases, the concentration of the additives, thesubstrate concentration and the enzyme activity on oligomers yield anddegree of oligomerization were monitored through a set of experiments.

[0271] B 1. Optimization of Aqueous and Non-aqueous Phase Ratio

[0272] The ratio of the non-aqueous phase volume to aqueous phase volumewas varied by changing the volumes of the two organic solvents in equalproportion while holding the aqueous phase volume constant. Substrates,antioxidant additives and enzyme were added to the aqueous phase. Thereactants and the enzyme were placed in a stirred reactor and allowed toincubate at 37° C. for 24 hours. The total oligomers yield wasdetermined gravimetrically, while the degree of oligomerization wasdetermined through RPLC analysis. Results of gravimetric determinationare represented in FIG. 44.

[0273] The results show that the oligomer yield increased with anincrease in total organic phase. However, the increase in yield wasrelatively small above an aqueous:organic ratio of 1:10. The effect ofphase ratio on the degree of oligomerization is shown in FIG. 45.Results show that while the total yields are lower (approximately15-50%) at the lower phase ratios, the degree of oligomerization ishigher and oligomers with up to 10 lysine residues can be readilyobtained. At higher phase ratios, the total oligomers yields aresignificantly greater (e.g., up to approximately 85%). The degree ofoligomerization was generally lower, however, as the predominantoligomers formed under these conditions contained three to five lysineresidues.

[0274] B 2. Optimization of Additives Concentration

[0275] The effect of the total additive concentration on the oligomersyield and degree of oligomerization was examined. At low additiveconcentrations (e.g., concentrations <1.5%), the overall oligomersyields were low (e.g., approximately 40%). An increase in additiveconcentration up to 2.5% led to an increase in the oligomers yield,however, concentration above this level did not lead to higher yields,FIG. 46. The concentration of the additives did not assert a pronouncedeffect on the distribution of lysine oligomers, FIG. 47.

[0276] B 3. Optimization of Incubation Period

[0277] The incubation period for the three-phase reaction system wasexamined through another set of experiments. The experiments wereconducted with three phase reaction mixtures consisting of aqueous phaseand total organic phases, the aqueous:organic phase ratio was set at1:9. The additive concentrations were varied. The incubation periodswere varied from 30 minutes to 30 hours. After each time period, totaloligomer yield and degree of oligomerization were examined. Results areshown in FIG. 48. The results indicate that the reaction reachescompletion in approximately six hours and nearly 90% of the initialsubstrate mass is converted into the oligomers. Longer incubationperiods did not lead to higher yields.

[0278] RPLC results showed that the longer incubation periods favoredoligomers with smaller lysine residues. The predominant residue after a24 hour incubation period was found to be (Lys)₄, FIG. 50.

[0279] The studies above demonstrate the effect of various parameters inthe three phase and two-phase systems and can be used to tailor thereaction to produce the required residue range and composition.

Example 11

[0280] Synthesis of Tryptophan Oligomers and HMB-Tryptophan Co-oligomers

[0281] A procedure similar to one used for the synthesis of Metoligomers and HMB-Met co-oligomers of Example 1 was employed for thesynthesis of tryptophan oligomers and HMB-Tryptophan co-oligomers.

[0282] Trp-OMe was dissolved in 4.5 mL of 2M citrate buffer along withthe EDTA and L-Cysteine. The pH was adjusted to 5 and 0.5 mL of papainsuspension was added to the mixture. The mixture was placed in a shakerfor three days and incubated. After three days, the enzyme was denaturedby heating the broth for a duration of 10 minutes at 80° C.

[0283] The broth was filtered to collect the precipitate. Alternatively,the broth could be centrifuged to collect the precipitate. The oligomerand co-oligomer precipitate was dissolved in DMF to separate them fromthe monomers which are relatively insoluble in the solvent. The solventwas evaporated and the precipitate was washed with water, followed byfreeze-drying the precipitate to obtain the dry oligomers andco-oligomers.

[0284] The procedure was also performed wherein L-Tyrosine Ethyl Esterwas substituted for Trp-OMe. The recipes for synthesis of differentoligomers and co-oligomers are summarized below in Table 5. TABLE 5Recipes for the Synthesis and Purification of L-Trp Homo-Oligomers andHMB-L-Trp Hetero-Co-Oligomers Jost/Trp-OEt Selvi/Trp-OMe Components MWMoles Wt Moles Wt Wt AA-ester 3 g 0.38 M 370 mg L-Cys.HCl.H₂O 175.6 100mM 0.1756 g 100 mM 0.0878 EDTA(anhyd) 292.0 10 mM 0.0292 g 10 mM 0.01461g Na Citrate 294.1 1 M 2.941  2 M 2.941 g Papain 21428 D 7*10⁻⁵ M 15 mg1.4*10⁻⁴ M 15 mg Volume 10 mL 5 mL pH 5.5 5

Example 12

[0285] Synthesis and Purification of Leucine, Phenylalanine, andTryptophan Oligomers

[0286] A procedure similar to one used for the synthesis of Metoligomers and HMB-Met co-oligomers of Example 1 was employed for thesynthesis and purification of leucine, phenylalanine, and tryptophanoligomers. An amino acid ester (e.g., 1 mmole) was dissolved in 10 mL of2M phosphate buffer solution at pH 7.5 containing 1 mM dithiothreitholand 5 mM EDTA. A papain suspension (e.g., 0.1 mmole) was added to thesolution. The solution was incubated for two days with continuousshaking, after which the precipitate formed was filtered and washed withwater several times to remove the free monomers. The precipitate wasdried in vacuo and then subjected to analysis.

[0287] The recipes for synthesis of different oligomers are summarizedbelow in Table 6. TABLE 6 Recipes for the Synthesis and Purification ofLeucine, Phenylalanine, and Tryptophan Oligomers Components Moles MW WtAA-OEt.HCl 0.1 mM Dithiothreithol 1 mM 154.2 0.00152 g EDTA 5 mM 292.20.01461 g Na₂HPO₄/NaH₂PO₄ 2 M (Phosphate Buffer) Volume 10 mL 10 mL

Example 13

[0288] A General Procedure for the Synthesis and Purification ofOligomers

[0289] L-amino acid ethyl ester or D, L-amino acid esters and racemicHMB ethyl ester were dissolved in buffer containing L-cysteine, EDTA andsodium citrate at pH 5.5 as detailed in Tables 5-9. The buffer pH wasset at 5.5 and 0.5 mL of papain suspension containing 15 mg of proteinwas added to the reaction broth. After incubation in a shaker for 24hours at 35° C., the enzyme was denatured by heating the broth to 80° C.for 10 minutes. The broth with denatured enzyme was then cooled to roomtemperature. The oligomer and co-oligomer precipitate obtained from thereaction was washed exhaustively with water to remove the monomers andthe washed precipitate was then freeze-dried. The freeze-dried oligomerand co-oligomer precipitate was solubilized in DMSO to form a 2 μg/μLsolution. To remove traces of free HMB-ester, HMB co-oligomers werere-precipitated by addition of distilled deionized water. The purifiedoligomers and co-oligomers were freeze dried. The oligomer andco-oligomer were dissolved in appropriate solvents or mixtures solution(DMSO and 1:1 acetonitrile:water mixture) for chemical characterizationwith liquid chromatography (LC) diode array detector, LC-electrospraymass spectrometry (ESI-MS), sonic spray ionization-MS (SSI-MS) andmatrix assisted desorption ionization-MS (MALDI-TOF).

[0290] Recipes for synthesis of different oligomers and co-oligomers aresummarized in Tables 7-11. TABLE 7 Recipes for the Synthesis andPurification of L-Met Oligomers and HMB-L-Met Co-Oligomers Components MWMoles Met HMB-Met L-AA-OEt.HCl 213.7 3 g 1.5 g DL-HMB-OEt 178 — 1.5 g L-175.6 100 mM 0.1756 g 0.1756 g Cys.HCl.H₂O EDTA (anhyd) 292.0 10 mM0.0292 g 0.0292 g Na Citrate 294.1 1 M 2.941 g 2.941 g Papain 21428D7*10⁻⁵ M 15 mg 15 mg Volume 10 mL 10 mL pH 5.5 5.5

[0291] TABLE 8 Recipes for the Synthesis and Purification of L-TyrOligomers and HMB-L-Tyr Co-Oligomers Components MW Moles Tyr HMB-TyrL-AA-OEt 933.36 mg 466.82 mg DL-HMB-OEt 178 — 338.2 mg L- 175.6 100 mM0.1756 g 0.1756 g Cys.HCl.H₂O EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 gNa Citrate 294.1 1 M 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mg Volume10 mL 10 mL pH 5.5 5.5

[0292] TABLE 9 Recipes for the Synthesis and Purification of L-LeuOligomers and HMB-L-Leu Co-Oligomers Components MW Moles Leu HMB-LeuL-AA-Oet 2.739 g 1.369 g DL-HMB-OEt 178 — 1.253 g L- 175.6 100 mM 0.1756g 0.1756 g Cys.HCl.H₂O EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 g NaCitrate 294.1 1 M 2.941 g 2.941 g Papain 21428 D 7 * 10⁻⁵ M 15 mg 15 mgVolume 10 mL 10 mL pH 5.5 5.5

[0293] TABLE 10 Recipes for the Synthesis and Purification of L-PheOligomers and HMB-L-Phe Co-Oligomers Components MW Moles Phe HMB-PheL-AA-Oet 1.1414 g 0.5707 g DL-HMB-OEt 178 — 0.4067 mg L- 175.6 100 mM0.1756 g 0.1756 g Cys.HCl.H₂O EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 gNa Citrate 294.1 1 M 2.941 g 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mgVolume 10 mL 10 mL pH 5.5 5.5

[0294] TABLE 11 Recipes for the Synthesis and Purification of L-TrpOligomers and HMB-L-Trp Co-Oligomers Components MW Moles Trp HMB-TrpL-AA-Oet 0.48 mg 0.480 g DL-HMB-OEt 178 — 0.480 mg L- 175.6 100 mM0.1756 g 0.1756 g Cys.HCl.H₂O EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 gNa Citrate 294.1 1 M 2.941 g 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mgVolume 10 mL 10 mL pH 5.5 5.5

Example 14

[0295] Enantioselectivity of Papain Catalyzed Oligomerization andCo-Oligomerization

[0296] A set of experiments was carried out to discernenantioselectivity of papain wherein oligomers and co-oligomers werecatalyzed from an amino acid and HMB. The experiments entailedenantioselective determination of the reactants (e.g., methionine andHMB co-oligomerization from enantiomeric mixtures of D, L-methionine andD, L-HMB and separation of supernatant and oligomer and co-oligomerprecipitates). The supernatant was filtered to remove suspended matter,and the precipitate was purified through repeated washing and DMSOback-extraction. The purified oligomer and co-oligomer precipitates werehydrolyzed with acid. The reactant solutions, supernatant, andhydrolyzates were subjected to enantioselective HPLC analysis. Theresults of the experiments indicated that catalytic co-oligomerizationof amino acids and HMB is enantioselective wherein only the L-HMB andthe L-amino acid isomers undergo oligomerization and co-oligomerization.

[0297] Papain Catalyzed Synthesis

[0298] The Met oligomers and HMB-Met co-oligomers were synthesizedthrough papain mediated enzymatic reactions at pH 5.5. The synthesisinvolved the following steps:

[0299] Racemic mixtures of both D, L-Met-OEt and D, L-HMB-OEt weredissolved in 4.5 mL of 2M citrate buffer along with EDTA and L-Cysteine.The pH was set to 5 and 0.5 mL of papain suspension was added to themixture. The mixture was incubated in a shaker for 3 days.

[0300] After the mixture was incubated in a shaker for 3 days, theenzyme (e.g., papain) was denatured by heating the mixture for 10minutes at 80° C.

[0301] The mixture was filtered and the precipitate collected.(Alternatively, the mixture may be centrifuged to separate theprecipitate from the supernatant).

[0302] The oligomers and co-oligomers were dissolved in DMF to separatethem from the monomers, which are relatively insoluble in DMF.

[0303] The solvent was evaporated and the precipitate washed with water,followed by freeze drying to obtain the dry oligomers and co-oligomers.

[0304] The recipe for synthesis of oligomers of α-amino acid isomershaving identical chiral configurations and co-oligomers formed from HMBisomers having a specific chiral configuration and α-amino acid isomershaving identical chiral configurations is summarized below in Table 12.TABLE 12 Recipe for the Synthesis and Purification of L-Met Oligomersand L-HMB-L-Met Co-Oligomers From Racemic Mixtures of D,L-MetOEt andD,L-HMB-OEt MetOEt/HMB-OEt Components MW Moles Wt AA-ester 3 gL-Cys.HCl.H₂O 175.6 100 mM 0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g NaCitrate 294.1 1 M 2.941 Papain 21428D 7*10⁻⁵ M 15 mg Volume 10 mL pH 5.5

[0305] Hydrolysis of Met Oligomers and Met/HMB Co-Oligomers

[0306] A 25 mg aliquot of purified oligomers and co-oligomers obtainedfrom the racemic Met-OEt and HMB-OEt was placed in 10 mL vials andhydrolyzed with 2 mL of 6.05(N) HCl at 110° C. for 24 hours. The clearsolutions obtained after hydrolysis were diluted with nanopure water andinjected in an LC equipped a diode array detector (DAD).

[0307] Enantioselective HPLC

[0308] Enantioselective HPLC analysis was carried out with a proline-Cubased column EC 250/4 Nucleosil Chiral 1 (Macherey-Nagel, Inc., Easton,Pa.). The mobile phase consisted of 0.5 mM cupric sulfate (pentahydrate)solution in nanopure water. Column oven temperature was maintained at60° C. Separated analytes were monitored with a UV absorbance DAD.

[0309] Results

[0310] The HPLC results showed that the Met-OEt and HMB-OEt reactantswere racemic mixtures that contained equal amounts of D- and L-Met ethylester enantiomers and D- and L-HMB ethyl ester enantiomers. See FIGS. 51and 52. Following oligomerization and co-oligomerization and separationof the precipitates, the supernatant was analyzed and found to containsignificantly higher concentrations of D-Met and D-HMB than L-Met andL-HMB. The relative concentrations of D-Met and L-Met were found to beapproximately 97%: <2.5% respectively. The relative concentrations ofD-HMB and L-HMB were found to be approximately 75% and 25% respectively.

[0311] The selective oligomerization and co-oligomerization of L-Met andL-HMB were observed in the results from the enantioselective HPLCanalysis. The analysis indicated that the oligomer and co-oligomerhydrolyzate was found to contain only the L-Met and the L-HMB isomers.See FIGS. 53 and 54. While FIG. 54 indicates the presence of a D-HMBisomer peak, when the precipitate was washed, dissolved in DMSO, andreprecipitated, the D-HMB isomer peak disappeared, indicating that theD-HMB present was initially absorbed to the oligomer and co-oligomerprecipitate, but was not covalently bonded within the oligomer andco-oligomer precipitate.

Example 15

[0312] Papain Catalyzed Synthesis of Lactic Acid-Amino Acid Oligomers

[0313] This example demonstrates the synthesis of co-oligomerscomprising lactic acid (an α-hydroxy carboxylic acid) with oligomers ofthe α-amino acids methionine, leucine, tyrosine, phenylalanine andtryptophan.

[0314] The experiment consisted of esterifying D,L-Lactic acid withacidified ethyl alcohol by refluxing it at 80° C. for 8 hrs to producelactic acid ethyl ester, which was used in each of the oligomerizationreactions. The oligomerization consisted of forming a mixture bydissolving each of the amino acid ethyl esters and the lactic acid ethylester in various amounts in a pH 5.5 buffer containing, L-cysteine, EDTAand sodium citrate as shown in the tables below. At the end of 24 hours,the mixture was heated to 80° C. for 10 mins to denature the enzyme. Thesupernatant was analyzed on RPLC to determine the yield of the reaction.The precipitate was washed thoroughly with water to obtain a monomer(amino acid and lactic acid) free product. The product was freeze driedand a small part of it was dissolved in DMSO and introduced into theESI-MS and the mass spectrum obtained was recorded.

[0315] 1. Lactic Acid-methionine Co-oligomerization

[0316] Lactic acid-methionine oligomers were synthesized using thegeneral procedure described above. The ingredients for theoligomerization reaction mixture consisted of the following: CompositionL-MetEE-HCI (g) 1.5 LAEE (g) 1.5 L-Cysteine (mg) 175.6 EDTA (mg) 29.2Sodium Citrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

[0317] The oligomerization produced an yield of 75% and the positive ionand negative ion spectra are reproduced in FIGS. 55A and 55Brespectively. The positive ion spectrum shows the presence of 2 dominantpeaks at 834 and 965. These peaks most probably represent thehomo-oligomers, ^(N)Met-(Met)₄-Met^(C)-OEt and^(N)Met-(Met)₅-Met^(C)-OEt respectively which are separated by therepeating methionine residue unit of mass 131.2.

[0318] The negative ion spectrum shows the presence of a series of peakseach separated by around 131 mass units. One set of peaks appear at 774and 905 and these most probably represent the deprotonated ions,^(N)LA-(Met)₄-Met^(C)-OEt and ^(N)LA-(Met)₅-Met^(C)-OEt respectively.Another set of ions, appear at 809 and 940 and these mot probablyrepresent the adducts of the above co-oligomers with the chloride ion.

[0319] 2. Lactic Acid-tyrosine Co-oligomerization

[0320] Lactic acid-tyrosine oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-TyrEE-HCI(mg) 466.8 LAEE (mg) 466.8 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 SodiumCitrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

[0321] A yield of 98% with respect to tyrosine was obtained. Thepositive ion and negative ion spectra are provided in FIG. 56A and 56Brespectively. The positive ion spectra shows the presence of a evenlyspaced sets of two peaks with each peak separated from the other by 28amu. This mass represents the difference in mass between C-terminal freeacid and ethyl ester. Each set of peaks is separated by a mass of 163units with is the repeating unit of the tyrosine residue. While one setappeared at 833, 996, 1159 and 1322 most probably representing theprotonated homo-oligomers of tyrosine namely, ^(N)Tyr-Tyr₃-Try^(C)-OH,^(N)Try-Tyr₄-Tyr^(C)-OH, ^(N)Tyr-Tyr₅-Tyr^(C)-OH and^(N)Tyr-Tyr₆-Tyr^(C)-OH, another set appeared 861, 1024 and 1187 andthese most probably represent the protonated forms ofNtyr-Tyr₃-Tyr^(C)-OEt,^(N)tyr-Tyr₄-Tyr^(C)-OEt and Ntyr-Tyr₅-Tyr^(C)-OEtrespectively. The negative ion spectrum reveals a number of peaks withsome of them forming a series separated by 163 units. One set appears at1096, 1259 and 1422 and these most probably represent the presence ofdeprotonated co-oligomer ions formed from^(N)LA-Tyr₅-Tyr^(C)-OEt,^(N)LA-Tyr₆-Tyr^(C)-OEt and^(N)LA-Tyr₇-Tyr^(C)-OEt respectively.

[0322] 3. Lactic Acid-leucine Co-oligomerization

[0323] Lactic acid-leucine oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-LeuEE-HCI(mg) 684.5 LAEE (mg) 684.5 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 SodiumCitrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

[0324] The oligomerization produced a yield of 40% with respect toleucine. The positive and negative ion specta are provided in FIGS. 57Aand 57B respectively. The positive ion spectrum has a pair of peaks at726 and 839 and these ions are separated by the repeating residue unitof leucine (113 amu) and they most probably represent the protonatedions of homo-oligomers, ^(N)Lleu-Leu₄-Leu^(C)-OEt and^(N)Leu-Leu₅-Leu^(C)-OEt respectively. Another ion appears at 698 andthis is most probably the protonated ion of the homo-oligomer^(N)Leu-Leu₄-Leu^(C)-OH. In addition to these peaks, another pair ofpeaks appear at 685 and 798 and these are again separated by therepeating unit of leucine and these most probably correspond to theprotonated forms of the co-oligomer peaks, ^(N)LA-Leu₄-Leu^(C)-OEt and^(N)LA-Leu₅-Leu^(C)-OEt respectively. The peaks corresponding to^(N)LA-Leu₄-Leu^(C)-OH⁺H⁺ and ^(N)LA-Leu₅-Leu^(C)-OEt+Na⁺ appear at 657and 821 respectively. The negative ion spectrum reveals a series ofpeaks each separated by 113 amu. These appear at 683, 796 and 909 andthey most probably represent the deprotonated forms of the co-oligomers^(N)LA-Leu₄-Leu^(C)-OEt-^(N)LA-Leu₅-Leu^(C)-OEt and^(N)LA-Leu₆-Leu^(C)-OEt respectively.

[0325] 4. Lactic Acid-tryptophan Co-oligomerization

[0326] Lactic acid-tryptophan oligomers were synthesized using thegeneral procedure described above. The ingredients for theoligomerization reaction mixture consisted of the following: CompositionL-TrpEE-HCI (mg) 480.0 LAEE (mg) 480.0 L-Cysteine (mg) 175.6 EDTA (mg)29.2 Sodium Citrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

[0327] An oligomerization yield of 94% was obtained with respect totryptophan. The positive ion and negative ion spectra are produced inFIGS. 58A and 58B respectively. A series of ions separated b 186 unitsat 419, 605, 791 and 977 appears in the positive ion spectrum. Theseindicate the presence of the protonated homo-oligomers of the form^(N)Trp_(n)-Trp^(c)-OEt with n 1 to 5. The ^(N)Trp-Trp-Trp^(C)-OH+H⁺ ionappears at 577. The ion at 700 most probably represent the loneco-oligomer peak of ^(N)LA-Trp₂-Trp^(C)-OEt+Na⁺. The negative ionspectrum shows the presence of deprotonated ^(N)Trp-Trp₂-Trp^(C)-OEt asthe base peak at 789. However, neither of the spectra does showexplicitly show the presence of co-oligomer peaks. Further work withLC-MS needs to be done to separate the oligomer from the co-oligomersand prove the presence/absence of the co-oligomers.

[0328] 5. Lactic Acid-phenylalanine Co-oligomerization

[0329] Lactic acid-phenylalanine oligomers were synthesized using thegeneral procedure described above. The ingredients for theoligomerization reaction mixture consisted of the following: CompositionL-PheEE-HCI (mg) 570.7 LA-IPA (mg) 570.7 L-Cysteine (mg) 175.6 EDTA (mg)29.2 Sodium Citrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

[0330] The lactic acid was esterified with iso-propyl alcohol and wasused along with the phenylalanine ethyl ester. An oligomerization yieldof 80% was obtained with respect to phenylalanine. The positive andnegative ion spectrum is provided in FIG. 59A and 59B respectively. Thepositive ion spectrum reveals the strong presence of sodiatedco-oligomers ions of ^(N)LA-(Phe)₃-Phe^(C)-OEt+Na⁺ and^(N)LA-(Phe)₄-Phe^(C)-OEt+Na⁺ at masses 730 and 877 respectively. Thesepeaks are separated by 147 units, which is the repeating residue unit ofphenylalanine. The absence of any oligomer peaks is due to fact that thephenylaline does not oligomerize without a N-terminal starter molecule.This points to the presence of LA at the N-terminal end of the oligomer.The presence of ethyl ester (and absence of the iso-propyl ester) at theC-terminal end also reinforces this result. The negative ion spectrumreveals two sets of peaks with each set separated by 147 units. Thesesets represent the deprotonated co-oligomer peaks to form^(N)LA-Phen-Phe^(C)-OEt with n having values of 3 and 4.

[0331] In view of the above, it will be seen that the several objects ofthe invention are achieved. As various changes could be made in theabove compositions and methods without departing from the scope of theinvention, it is intended that all matter contained in the abovedescription be interpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. A process for the preparation of an oligomer, theprocess comprising: forming a reaction mixture comprising an enzyme, andan enantiomeric mixture of an α-amino acid or a derivative thereof; andforming an oligomer from the reaction mixture, said oligomerincorporating one of the members of the enantiomeric mixture of theα-amino acid or derivative thereof in preference to the other member. 2.A process as set forth in claim 1, wherein the enantiomeric mixture is aracemic mixture.
 3. A process as set forth in claim 1, wherein theoligomer comprises an α-amino acid or a derivative thereof comprising anoligomer of two or more α-amino acid monomers.
 4. A process as set forthin claim 3, wherein the α-amino acid monomers comprise L-α-amino acidmonomers.
 5. A process as set forth in claim 1, wherein the reactionmixture further comprises an α-hydroxy carboxylic acid or a derivativethereof and the formed oligomer comprises an oligomer of two or moreα-amino acid monomers bonded to a residue of the α-hydroxy carboxylicacid or a derivative thereof by an amide or ester linkage.
 6. A processas set forth in claim 5 wherein said α-hydroxy carboxylic acid or aderivative thereof is present in said reaction mixture in anenantiomeric mixture.
 7. A process as set forth in claim 6 wherein oneα-hydroxy carboxylic acid enantiomer of the enantiomeric mixture isbonded to the oligomer in preference to the other enantiomer of saidenantiomeric mixture.
 8. A process as set forth in claim 6 wherein theα-hydroxy carboxylic acid comprises 2-hydroxy-4-(methylthio)butyric acidor a derivative thereof.
 9. A process as set forth in claim 8, whereinthe α-amino acid monomers in the oligomer comprise L-α-amino acidmonomers.
 10. A process as set forth in claim 1 wherein the α-amino acidis selected from the group consisting of methionine, lysine, or aderivative thereof.
 11. A process as set forth in claim 1 wherein saidenantiomeric mixture of α-amino acids comprises methionine and lysine.12. A process as set forth in claim 1 wherein the enzyme comprises aprotease.
 13. A process as set forth in claim 12 wherein the enzymecomprises a protease selected from the group consisting of papain,bromelain, cathepsin s, cathepsin b, capthesin c, and substilisin.
 14. Aprocess as set forth in claim 1 wherein the reaction mixture comprisesan aqueous solution.
 15. A process as set forth in claim 1 wherein thereaction mixture comprises an aqueous phase and an organic solventphase.
 16. A process as set forth in claim 1 wherein the reactionmixture comprises an α-hydroxy carboxylic acid derivative comprising afree acid, an acid halide, an amide, an ester or an anhydride.
 17. Aprocess as set forth in claim 1 wherein the reaction mixture comprisesan α-amino acid derivative comprising a free acid, an acid halide, anamide, an ester or an anhydride.
 18. A composition comprising a residueof an α-hydroxy carboxylic acid bonded to a peptide by an amide linkage,said peptide comprising two or more α-amino acid residues, each of saidα-amino acids being independently selected from the group consisting ofα-amino acids.
 19. A composition as set forth in claim 18 wherein morethan 50% of the α-amino acid residues in the peptide are of identicalchirality.
 20. A composition as set forth in claim 19 whereinessentially all of the α-amino acid residues in the peptide are ofidentical chirality.
 21. A composition comprising a residue of anα-hydroxy carboxylic acid bonded to a peptide by an ester linkage, saidpeptide comprising two or more α-amino acid residues, each of saidα-amino acids being independently selected from the group consisting ofα-amino acids.
 22. A composition as set forth in claim 21 wherein morethan 50% of the α-amino acid residues in the peptide are of identicalchirality.
 23. A composition as set forth in claim 22 whereinessentially all of the α-amino acid residues in the peptide are ofidentical chirality.
 24. A composition comprising:

R¹ is hydrogen, hydrocarbyl or substituted hydrocarbyl, R² is hydrogen,hydrocarbyl, substituted hydrocarbyl, or a hydroxy protecting group, R³is hydrogen, hydrocarbyl or substituted hydrocarbyl, each AA is theresidue of an α-amino acid or derivative thereof wherein greater thanone-half of the AA residues are derived from α-amino acids orderivatives thereof having the an identical chiral configuration, and nis at least
 2. 25. The composition of claim 24 wherein R¹ isCH₃SCH₂CH₂—.
 26. The composition of claim 25 wherein R² is H.
 27. Thecomposition of claim 24 wherein R² is H.
 28. The composition of claim 24wherein n is at least 4 and no more than 12 and each AA is methionine.29. The composition of claim 24 wherein n is at least 3 and no more than5 and each AA is selected from the group consisting of methionine andlysine.
 30. The composition of claim 24 wherein n is at least 2 and nomore than 10 and each AA is selected from the group consisting ofmethionine and lysine.
 31. A process of providing a food ration to ananimal comprising providing the composition of claim 24 to the animalwherein the method of administration is selected from the groupconsisting of oral administration, eye spray, placement in ear,placement in nasal cavity, bucchal administration, sublingualadministration, rectal administration and injection.
 32. A process asset forth in claim 31 wherein the composition is orally administered tothe animals.
 33. A process as set forth in claim 32 wherein the animalis selected from the group consisting of ruminants, swine, poultry, andaquatic animals.
 34. A process as set forth in claim 33 wherein theruminant is a dairy cow or beef cattle.
 35. A process as set forth inclaim 34 wherein the cow is a lactating dairy cow.
 36. A process as setforth in claim 33 wherein the aquatic animal is a fish or crustacean.37. A process as set forth in claim 36 wherein the fish is selected fromthe group consisting of freshwater and salt water fish.
 38. A process asset forth in claim 37 wherein the freshwater and salt water fish areselected from the group consisting of carp, trout, catfish, bass, seabass, cod, and salmon.
 39. A process as set forth in claim 36 whereinthe crustaceans are selected from the group consisting of shrimp, crabs,lobster, and freshwater shrimp.
 40. A process for providing a foodration to an animal, the process comprising: providing an oligomer orco-oligomer composition prepared from a mixture containing an enzyme, anα-amino acid or a derivative thereof, and optionally, an α-hydroxycarboxylic acid or a derivative thereof, wherein the method ofadministration is selected from the group consisting of oraladministration, eye spray, placement in ear, placement in nasal cavity,bucchal administration, sublingual administration, rectal administrationand injection.
 41. A process as set forth in claim 40 wherein the animalis selected from the group consisting of ruminants, swine, poultry andaquatic animals.
 42. A process as set forth in claim 41 wherein theruminant is a dairy cow or beef cattle.
 43. A process as set forth inclaim 42 wherein the cow is a lactating dairy cow.
 44. A process as setforth in claim 41 wherein the aquatic animal is a fish or crustacean.45. A process as set forth in claim 44 wherein the fish is selected fromthe group consisting of freshwater and salt water fish.
 46. A process asset forth in claim 45 wherein the freshwater and salt water fish areselected from the group consisting of carp, trout, catfish, bass, seabass, cod, and salmon.
 47. A process as set forth in claim 44 whereinthe crustaceans are selected from the group consisting of shrimp, crabs,lobster, and freshwater shrimp.
 48. An orally administered dietarysupplement comprising a vitamin, a mineral or a nutrient coated with anoligomeric coating, said coating comprising a residue of an α-hydroxycarboxylic acid bonded to a peptide by an amide linkage, said peptidecomprising two or more α-amino acid residues, each of said α-amino acidsbeing independently selected from the group consisting of α-amino acids.49. A process for providing an animal with a dietary supplementcomprising a vitamin, mineral or nutrient, the process comprising:coating said vitamin, mineral or nutrient with a composition to form adietary supplement, said composition comprising a residue of anα-hydroxy carboxylic acid bonded to a peptide by an amide linkage, saidpeptide comprising two or more α-amino acid residues, each of saidα-amino acids being independently selected from the group consisting ofα-amino acids; and orally administering the dietary supplement to theanimal.
 50. A process as set forth in claim 49 wherein the peptidecomprises methionine.
 51. A process as set forth in claim 49 wherein theanimal is selected from the group consisting of ruminants, swine,poultry, and aquatic animals.
 52. A process as set forth in claim 51wherein the ruminant comprises a dairy cow or beef cattle.
 53. A processas set forth in claim 52 wherein the ruminant comprises a lactatingdairy cow.
 54. A process as set forth in claim 51 wherein the aquaticanimal comprises a fish or crustacean.
 55. A process as set forth inclaim 54 wherein the fish is selected from the group consisting offreshwater and salt water fish.
 56. A process as set forth in claim 55wherein the freshwater and salt water fish are selected from the groupconsisting of carp, trout, catfish, bass, sea bass, cod, and salmon. 57.A process as set forth in claim 54 wherein the crustaceans are selectedfrom the group consisting of shrimp, crabs, lobster, and freshwatershrimp.
 58. A process for providing an animal with a dietary supplementcomprising a vitamin, mineral or nutrient, the process comprising:preparing a mixture comprising an enzyme and at least one α-amino acid,each α-amino acid being present in the mixture as a free acid, acidhalide, amide, ester or anhydride independently of the other, forming anα-amino acid oligomer in the mixture, coating said vitamin, mineral ornutrient with the α-amino acid oligomer to form an α-amino acid oligomercoated dietary supplement, and orally administering the dietarysupplement to the animal.
 59. A process as set forth in claim 58 whereinthe animal comprises an aquatic animal selected from the groupconsisting of shrimp, carp, and trout.
 60. A process as set forth inclaim 59, wherein the α-amino acid oligomeric coating comprisesmethionine.
 61. A process for purifying an α-hydroxy carboxylic acidenantiomer or derivative thereof from an enantiomeric mixture, theprocess comprising: forming a reaction mixture comprising (i) anenantioselective enzyme, (ii) an enantiomeric mixture of an α-hydroxycarboxylic acid or a derivative thereof, and (iii) an enantiomericmixture of an α-amino acid or a derivative thereof; forming a reactionproduct from the reaction mixture, the reaction product comprising (i)an oligomer having a first α-hydroxy carboxylic acid enantiomer of theenantiomeric mixture incorporated therein in preference to the secondenantiomer, and (ii) unreacted second enantiomer; and separating theoligomer and unreacted second enantiomer from the reaction product andeach other.
 62. A process as set forth in claim 61, wherein theenantioselective enzyme promotes an esterification reaction.
 63. Aprocess as set forth in claim 62, wherein the enantioselective enzyme isa lipase enzyme.
 64. A process as set forth in claim 61, wherein thefirst enantiomer is recovered from the separated oligomer by hydrolyzingthe oligomer with acid and separating the first α-hydroxy carboxylicacid enantiomer or derivative thereof from other hydrolyzates.
 65. Aprocess as set forth in claim 64, wherein the first α-hydroxy carboxylicacid enantiomer or derivative thereof is separated by subjection toenantioselective chromatography.
 66. A process as set forth in claim 61,wherein the second unreacted α-hydroxy carboxylic acid enantiomer orderivative thereof is recovered from the reaction mixture by rotaryevaporation.
 67. A process as set forth in claim 61, wherein theα-hydroxy carboxylic acid comprises 2-hydroxy-4-(methylthio)butyric acidor a derivative thereof.
 68. A process as set forth in claim 61 whereinthe process further comprises recovering the first enantiomer from saidseparated oligomer by by hydrolyzing the oligomer with acid andseparating the first α-hydroxy carboxylic acid enantiomer or derivativethereof from other hydrolyzates; and converting a fraction of therecovered first enantiomer to the stereochemical form of the secondenantiomer thereby forming an enantiomeric mixture comprising the firstand second enantiomers.
 69. A process as set forth in claim 61 whereinthe process further comprises recovering the unreacted second enantiomerfrom the reaction product; and converting a fraction of the separatedunreacted second enantiomer to the stereochemical form of the firstenantiomer thereby forming an enantiomeric mixture comprising the firstand second enantiomers, at least a portion of the first enantiomer inthe enantiomeric mixture being derived from separated unreacted secondenantiomer.
 70. A process as set forth in claim 69 wherein theenantiomeric mixture formed from the recovered second enantiomer isrecycled for re-use in the formation of a reaction mixture.
 71. Aprocess as set forth in claim 69, wherein the enzyme is removed from thereaction mixture and recycled.
 72. A process as set forth in claim 71,wherein the enzyme is removed from the reaction mixture by sizeexclusion chromatography.
 73. A process as set forth in claim 69 whereinthe second unreacted α-amino acid enantiomers or derivatives thereof areseparated from the reaction mixture by rotary evaporation.
 74. A processfor purifying an enantiomeric mixture of α-amino acid or derivativethereof, the process comprising: forming a reaction mixture comprising(i) an enzyme, (ii) an enantiomeric mixture of α-amino acid or aderivative thereof, and (iii) an α-hydroxy carboxylic acid or aderivative thereof; forming a reaction product from the reactionmixture, the reaction product comprising (i) an oligomer incorporating afirst member of the enantiomeric mixture of α-amino acid or derivativethereof in preference to a second enantiomer of the enantiomeric mixtureor derivative thereof, and (ii) unreacted second enantiomer; andseparating the oligomer and unreacted second enantiomer from thereaction product and each other.
 75. A process as set forth in claim 74,wherein the first enantiomer is recovered from the separated oligomer byhydrolyzing the oligomer with acid and separating the α-amino acidenantiomer or derivative thereof from other hydrolyzates.
 76. A processas set forth in claim 75, wherein the α-amino acid enantiomer orderivative thereof is separated by subjection to enantioselectivechromatography.
 77. A process as set forth in claim 74, wherein theunreacted α-amino acid enantiomer or derivative thereof is recoveredfrom the reaction mixture by rotary evaporation.
 78. A process as setforth in claim 74, wherein the α-amino acid is selected from the groupconsisting of methionine and lysine.
 79. A process as set forth in claim74, wherein the α-hydroxy carboxylic acid comprises2-hydroxy-4-(methylthio)butyric acid or a derivative thereof.
 80. Aprocess for purifying an α-amino acid enantiomer or derivative thereofin an enantiomeric mixture, the process comprising: forming a reactionmixture comprising (i) an enantioselective enzyme and (ii) anenantiomeric mixture of an α-amino acid or derivative thereof; forming apeptide reaction product mixture comprising (i) an oligomer or aco-oligomer having a first enantiomer of the enantiomeric mixtureincorporated therein in preference to the second enantiomer of theenantiomeric mixture, and (ii) unreacted second enantiomer; andseparating the oligomer or co-oligomer and unreacted second enantiomerfrom the reaction product mixture and each other
 81. A process as setforth in claim 80, wherein the first enantiomer is recovered from theseparated oligomer or co-oligomer by hydrolyzing the oligomer orco-oligomer with acid and separating the α-amino acid enantiomer orderivative thereof from other hydrolyzates.
 82. A process as set forthin claim 81, wherein the first enantiomer or derivative thereof isseparated by subjection to enantioselective chromatography.
 83. Aprocess as set forth in claim 80, wherein the unreacted α-amino acidenantiomer or derivative thereof is recovered from the reaction mixtureby rotary evaporation.
 84. A process as set forth in claim 80 whereinthe α-hydroxy carboxylic acid derivative is a free acid, acid halide,amide, ester or anhydride.
 85. A process as set forth in claim 84wherein the α-amino acid derivative is a free acid, acid halide, amide,ester or anhydride.